Secondary literature sources for PYNP_C
The following references were automatically generated.
- Nishitani Y et al.
- Structure analysis of archaeal AMP phosphorylase reveals two unique modes of dimerization.
- J Mol Biol. 2013; 425: 2709-21
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AMP phosphorylase (AMPpase) catalyzes the initial reaction in a novel AMP metabolic pathway recently found in archaea, converting AMP and phosphate into adenine and ribose 1,5-bisphosphate. Gel-filtration chromatography revealed that AMPpase from Thermococcus kodakarensis (Tk-AMPpase) forms an exceptionally large macromolecular structure (>40-mers) in solution. To investigate its unique multimerization feature, we determined the first crystal structures of Tk-AMPpase, in the apo-form and in complex with substrates. Structures of two truncated forms of Tk-AMPpase (Tk-AMPpaseDeltaN84 and Tk-AMPpaseDeltaC10) clarified that this multimerization is achieved by two dimer interfaces within a single molecule: one by the central domain and the other by the C-terminal domain, which consists of an unexpected domain-swapping interaction. The N-terminal domain, characteristic of archaeal enzymes, is essential for enzymatic activity, participating in multimerization as well as domain closure of the active site upon substrate binding. Moreover, biochemical analysis demonstrated that the macromolecular assembly of Tk-AMPpase contributes to its high thermostability, essential for an enzyme from a hyperthermophile. Our findings unveil a unique archaeal nucleotide phosphorylase that is distinct in both function and structure from previously known members of the nucleoside phosphorylase II family.
- Lashkov AA et al.
- X-ray structure of Salmonella typhimurium uridine phosphorylase complexed with 5-fluorouracil and molecular modelling of the complex of 5-fluorouracil with uridine phosphorylase from Vibrio cholerae.
- Acta Crystallogr D Biol Crystallogr. 2012; 68: 968-74
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Uridine phosphorylase (UPh), which is a key enzyme in the reutilization pathway of pyrimidine nucleoside metabolism, is a validated target for the treatment of infectious diseases and cancer. A detailed analysis of the interactions of UPh with the therapeutic ligand 5-fluorouracil (5-FUra) is important for the rational design of pharmacological inhibitors of these enzymes in prokaryotes and eukaryotes. Expanding on the preliminary analysis of the spatial organization of the active centre of UPh from the pathogenic bacterium Salmonella typhimurium (StUPh) in complex with 5-FUra [Lashkov et al. (2009), Acta Cryst. F65, 601-603], the X-ray structure of the StUPh-5-FUra complex was analysed at atomic resolution and an in silico model of the complex formed by the drug with UPh from Vibrio cholerae (VchUPh) was generated. These results should be considered in the design of selective inhibitors of UPhs from various species.
- Vande Voorde J, Gago F, Vrancken K, Liekens S, Balzarini J
- Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues.
- Biochem J. 2012; 445: 113-23
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In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 muM) and deoxyuridine (Km=578 muM), it prefers uridine (Km=92 muM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.
- Marco-Marin C, Gil-Ortiz F, Perez-Arellano I, Cervera J, Fita I, Rubio V
- A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase.
- J Mol Biol. 2007; 367: 1431-46
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Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 A and 2.5 A resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha(3)beta(8)alpha(4) sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the beta sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta(5)beta(4) sandwich fold and three alpha helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement for free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P. The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres, which is possibly involved in the cooperativity for glutamate.
- Cui Z, Maruyama Y, Mikami B, Hashimoto W, Murata K
- Crystal structure of glycoside hydrolase family 78 alpha-L-Rhamnosidase from Bacillus sp. GL1.
- J Mol Biol. 2007; 374: 384-98
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alpha-L-Rhamnosidase (EC 220.127.116.11) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debittering agent for citrus fruit in the food and beverage industries through the release of rhamnose from plant glycoside, naringin. The X-ray crystal structure of RhaB was determined by single-wavelength anomalous diffraction using a selenomethionine derivative and refined at 1.9 A resolution with a final R-factor of 18.2%. As is seen in the homodimeric form of the active enzyme, the structure of RhaB in crystal packing is a homodimer containing 1908 amino acids (residues 3-956), 43 glycerol molecules, four calcium ions, and 1755 water molecules. The overall structure consists of five domains, four of which are beta-sandwich structures designated as domains N, D1, D2, and C, and an (alpha/alpha)(6)-barrel structure designated as domain A. Structural comparison by DALI showed that RhaB shares its highest level of structural similarity with chitobiose phosphorylase (Z score of 25.3). The structure of RhaB in complex with the reaction product rhamnose (inhibitor constant, K(i)=1.8 mM) was also determined and refined at 2.1 A with a final R-factor of 19.5%. Rhamnose is bound to the deep cleft of the (alpha/alpha)(6)-barrel domain, as is seen in the clan-L GHs. Several negatively charged residues, such as Asp567, Glu572, Asp579, and Glu841, conserved in GH family 78 enzymes, interact with rhamnose, and RhaB mutants of these residues have drastically reduced enzyme activity, indicating that the residues are crucial for enzyme catalysis and/or substrate binding. To our knowledge, this is the first report on the determination of the crystal structure of alpha-L-rhamnosidase and identification of its clan-L (alpha/alpha)(6)-barrel as a catalytic domain.
- Gao XF, Huang XR, Sun CC
- Role of each residue in catalysis in the active site of pyrimidine nucleoside phosphorylase from Bacillus subtilis: a hybrid QM/MM study.
- J Struct Biol. 2006; 154: 20-6
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Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway. We have built a model of a closed active conformation of the three-dimensional structure of PYNP from Bacillus subtilis. Using docking, molecular dynamics, and hybrid quantum-mechanical/molecular-mechanical methods to study the reaction mechanics between PYNP and a substrate, we identified the role of each residue in the active site during the entire catalytic process. The results indicate that the function of His(82), Arg(169), and Lys(188) is to stabilize the uridine in a high-energy conformation by means of electrostatic interactions and that these residues are involved in catalysis. In addition, the function of Asp(162) is likely to activate Lys(188) for phosphorolytic catalysis through polarization effects.
- Brux C et al.
- The structure of an inverting GH43 beta-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues.
- J Mol Biol. 2006; 359: 97-109
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beta-D-Xylosidases are glycoside hydrolases that catalyze the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicellulose. Here we describe the enzyme-substrate crystal structure of an inverting family 43 beta-xylosidase, from Geobacillus stearothermophilus T-6 (XynB3). Each XynB3 monomeric subunit is organized in two domains: an N-terminal five-bladed beta-propeller catalytic domain, and a beta-sandwich domain. The active site possesses a pocket topology, which is mainly constructed from the beta-propeller domain residues, and is closed on one side by a loop that originates from the beta-sandwich domain. This loop restricts the length of xylose units that can enter the active site, consistent with the exo mode of action of the enzyme. Structures of the enzyme-substrate (xylobiose) complex provide insights into the role of the three catalytic residues. The xylose moiety at the -1 subsite is held by a large number of hydrogen bonds, whereas only one hydroxyl of the xylose unit at the +1 subsite can create hydrogen bonds with the enzyme. The general base, Asp15, is located on the alpha-side of the -1 xylose sugar ring, 5.2 Angstroms from the anomeric carbon. This location enables it to activate a water molecule for a single-displacement attack on the anomeric carbon, resulting in inversion of the anomeric configuration. Glu187, the general acid, is 2.4 Angstroms from the glycosidic oxygen atom and can protonate the leaving aglycon. The third catalytic carboxylic acid, Asp128, is 4 Angstroms from the general acid; modulating its pK(a) and keeping it in the correct orientation relative to the substrate. In addition, Asp128 plays an important role in substrate binding via the 2-O of the glycon, which is important for the transition-state stabilization. Taken together, these key roles explain why Asp128 is an invariant among all five-bladed beta-propeller glycoside hydrolases.
- Buddha MR, Crane BR
- Structures of tryptophanyl-tRNA synthetase II from Deinococcus radiodurans bound to ATP and tryptophan. Insight into subunit cooperativity and domain motions linked to catalysis.
- J Biol Chem. 2005; 280: 31965-73
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An auxiliary tryptophanyl tRNA synthetase (drTrpRS II) that interacts with nitric-oxide synthase in the radiation-resistant bacterium Deinococcus radiodurans charges tRNA with tryptophan and 4-nitrotryptophan, a specific nitration product of nitric-oxide synthase. Crystal structures of drTrpRS II, empty of ligands or bound to either Trp or ATP, reveal that drTrpRS II has an overall structure similar to standard bacterial TrpRSs but undergoes smaller amplitude motions of the helical tRNA anti-codon binding (TAB) domain on binding substrates. TAB domain loop conformations that more closely resemble those of human TrpRS than those of Bacillus stearothermophilus TrpRS (bsTrpRS) indicate different modes of tRNA recognition by subclasses of bacterial TrpRSs. A compact state of drTrpRS II binds ATP, from which only minimal TAB domain movement is necessary to bring nucleotide in contact with Trp. However, the signature KMSKS loop of class I synthetases does not completely engage the ATP phosphates, and the adenine ring is not well ordered in the absence of Trp. Thus, progression of the KMSKS loop to a high energy conformation that stages acyl-adenylation requires binding of both substrates. In an asymmetric drTrpRS II dimer, the closed subunit binds ATP, whereas the open subunit binds Trp. A crystallographically symmetric dimer binds no ligands. Half-site reactivity for Trp binding is confirmed by thermodynamic measurements and explained by an asymmetric shift of the dimer interface toward the occupied active site. Upon Trp binding, Asp68 propagates structural changes between subunits by switching its hydrogen bonding partner from dimer interface residue Tyr139 to active site residue Arg30. Since TrpRS IIs are resistant to inhibitors of standard TrpRSs, and pathogens contain drTrpRS II homologs, the structure of drTrpRS II provides a framework for the design of potentially useful antibiotics.
- Yu Y et al.
- Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment: insights into substrate recognition, tRNA binding, and angiogenesis activity.
- J Biol Chem. 2004; 279: 8378-88
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Human tryptophanyl-tRNA synthetase (hTrpRS) produces a full-length and three N terminus-truncated forms through alternative splicing and proteolysis. The shortest fragment that contains the aminoacylation catalytic fragment (T2-hTrpRS) exhibits the most potent angiostatic activity. We report here the crystal structure of T2-hTrpRS at 2.5 A resolution, which was solved using the multi-wavelength anomalous diffraction method. T2-hTrpRS shares a very low sequence homology of 22% with Bacillus stearothermophilus TrpRS (bTrpRS); however, their overall structures are strikingly similar. Structural comparison of T2-hTrpRS with bTrpRS reveals substantial structural differences in the substrate-binding pocket and at the entrance to the pocket that play important roles in substrate binding and tRNA binding. T2-hTrpRS has a wide opening to the active site and adopts a compact conformation similar to the closed conformation of bTrpRS. These results suggest that mammalian and bacterial TrpRSs might use different mechanisms to recognize the substrate. Modeling studies indicate that tRNA binds with the dimeric enzyme and interacts primarily with the connective polypeptide 1 of hTrpRS via its acceptor arm and the alpha-helical domain of hTrpRS via its anticodon loop. Our results also suggest that the angiostatic activity is likely located at the alpha-helical domain, which resembles the short chain cytokines.
- Mendieta J et al.
- Role of histidine-85 in the catalytic mechanism of thymidine phosphorylase as assessed by targeted molecular dynamics simulations and quantum mechanical calculations.
- Biochemistry. 2004; 43: 405-14
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The structural changes taking place in the enzyme thymidine phosphorylase (TPase, also known as PD-ECGF) that are required to achieve catalytic competence upon binding thymidine and phosphate have been simulated by means of targeted molecular dynamics (tMD). The hinge regions were characterized by structural homology comparisons with pyrimidine nucleoside phosphorylase, whose X-ray structure has been solved both in a closed and in an open form. The rearrangement of residues around the substrate that was observed during the tMD trajectory suggested that His-85 could be playing an important role in the catalytic mechanism. A quantum mechanical study of the reaction in the presence of the most relevant active site residues was then performed at the semiempirical level. The results revealed that His-85 could be involved in the protonation of the pyrimidine base at the O2 position to yield the enol tautomer of the base. To establish the role of this oxygen atom in the reaction, ground states, transition states, and final products were studied using higher level ab initio methods starting from both thymidine and 2-thiothymidine as alternative substrates. Comparison of both transition states showed that replacing the oxygen at position 2 of the pyrimidine base by sulfur should accelerate the reaction rate. Consistent with this result, 2-thiothymidine was shown to be a better substrate for TPase than the natural substrate, thymidine. For simulating the final step of the reaction, tMD simulations were used to study domain opening upon product formation considering both the enol and keto tautomers of thymine. Product release from the enzyme was easiest in the simulation that incorporated the keto tautomer of thymine, suggesting that the enol intermediate spontaneously tautomerizes back to the more energetically stable keto form. These results highlight a previously unreported role for His-85 in the catalytic mechanism of TPase and can have important implications for the design of novel TPase inhibitors.
- Bzowska A et al.
- Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis.
- J Mol Biol. 2004; 342: 1015-32
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The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.
- Caradoc-Davies TT, Cutfield SM, Lamont IL, Cutfield JF
- Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium.
- J Mol Biol. 2004; 337: 337-54
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Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.
- Ceccarelli C et al.
- Crystal structure and amide H/D exchange of binary complexes of alcohol dehydrogenase from Bacillus stearothermophilus: insight into thermostability and cofactor binding.
- Biochemistry. 2004; 43: 5266-77
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The crystal structure of NAD(+)-dependent alcohol dehydrogenase from Bacillus stearothermophilus strain LLD-R (htADH) was determined using X-ray diffraction data at a resolution of 2.35 A. The structure of homotetrameric htADH is highly homologous to those of bacterial and archaeal homotetrameric alcohol dehydrogenases (ADHs) and also to the mammalian dimeric ADHs. There is one catalytic zinc atom and one structural zinc atom per enzyme subunit. The enzyme was crystallized as a binary complex lacking the nicotinamide adenine dinucleotide (NAD(+)) cofactor but including a zinc-coordinated substrate analogue trifluoroethanol. The binary complex structure is in an open conformation similar to ADH structures without the bound cofactor. Features important for the thermostability of htADH are suggested by a comparison with a homologous mesophilic enzyme (55% identity), NAD(+)-dependent alcohol dehydrogenase from Escherichia coli. To gain insight into the conformational change triggered by NAD(+) binding, amide hydrogen-deuterium exchange of htADH, in the presence and absence of NAD(+), was studied by HPLC-coupled electrospray mass spectrometry. When the deuteron incorporation of the protein-derived peptides was analyzed, it was found that 9 of 21 peptides show some decrease in the level of deuteron incorporation upon NAD(+) binding, and another 4 peptides display slower exchange rates. With one exception (peptide number 8), none of the peptides that are altered by bound NAD(+) are in contact with the alcohol-substrate-binding pocket. Furthermore, peptides 5 and 8, which are located outside the NAD(+)-binding pocket, are notable by displaying changes upon NAD(+) binding. This suggests that the transition from the open to the closed conformation caused by cofactor binding has some long-range effects on the protein structure and dynamics.
- Luic M, Koellner G, Yokomatsu T, Shibuya S, Bzowska A
- Calf spleen purine-nucleoside phosphorylase: crystal structure of the binary complex with a potent multisubstrate analogue inhibitor.
- Acta Crystallogr D Biol Crystallogr. 2004; 60: 1417-24
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Purine-nucleoside phosphorylase (PNP) deficiency in humans leads to inhibition of the T-cell response. Potent membrane-permeable inhibitors of this enzyme are therefore considered to be potential immunosuppressive agents. The binary complex of the trimeric calf spleen phosphorylase, which is highly homologous to human PNP, with the potent ground-state analogue inhibitor 9-(5,5-difluoro-5-phosphonopentyl)guanine (DFPP-G) was crystallized in the cubic space group P2(1)3, with unit-cell parameter a = 93.183 A and one monomer per asymmetric unit. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL Outstation, DESY, Hamburg, station X13). The crystal structure was refined to a resolution of 2.2 A and R and Rfree values of 19.1 and 24.2%, respectively. The crystal structure confirms that DFPP-G acts as a multisubstrate analogue inhibitor as it binds to both nucleoside- and phosphate-binding sites. The structure also provides the answers to some questions regarding the substrate specificity and molecular mechanism of trimeric PNPs. The wide access to the active-site pocket that was observed in the reported structure as a result of the flexibility or disorder of two loops (residues 60-65 and 251-266) strongly supports the random binding of substrates. The putative hydrogen bonds identified in the base-binding site indicate that N1-H and not O6 of the purine base defines the specificity of trimeric PNPs. This is confirmed by the fact that the contact of guanine O6 with Asn243 Odelta1 is not a direct contact but is mediated by a water molecule. Participation of Arg84 in the binding of the phosphonate group experimentally verifies the previous suggestion [Blackburn & Kent (1986), J. Chem. Soc. Perkin Trans. I, pp. 913-917; Halazy et al. (1991), J. Am. Chem. Soc. 113, 315-317] that fluorination of alkylphosphonates yields compounds with properties that suitably resemble those of phosphate esters and in turn leads to optimized interactions of such analogues with the phosphate-binding site residues. DFPP-G shows a Ki(app) in the nanomolar range towards calf and human PNPs. To date, no high-resolution X-ray structures of these enzymes with such potent ground-state analogue inhibitors have been available in the Protein Data Bank. The present structure may thus be used in the rational structure-based design of new PNP inhibitors with potential medical applications.
- Retailleau P et al.
- Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed, pre-transition-state conformations.
- J Mol Biol. 2003; 325: 39-63
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Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.
- Hondoh H, Kuriki T, Matsuura Y
- Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase.
- J Mol Biol. 2003; 326: 177-88
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Crystal structures of Bacillus stearothermophilus TRS40 neopullulanase and its complexes with panose, maltotetraose and isopanose were determined at resolutions of 1.9, 2.4, 2.8 and 3.2A, respectively. Since the latter two carbohydrates are substrates of this enzyme, a deactivated mutant at the catalytic residue Glu357-->Gln was used for complex crystallization. The structures were refined at accuracies with r.m.s. deviations of bond lengths and bond angles ranging from 0.005A to 0.008A and 1.3 degrees to 1.4 degrees, respectively. The active enzyme forms a dimer in the crystalline state and in solution. The monomer enzyme is composed of four domains, N, A, B and C, and has a (beta/alpha)(8)-barrel in domain A. The active site lies between domain A and domain N from the other monomer. The results show that dimer formation makes the active-site cleft narrower than those of ordinary alpha-amylases, which may contribute to the unique substrate specificity of this enzyme toward both alpha-1,4 and alpha-1,6-glucosidic linkages. This specificity may be influenced by the subsite structure. Only subsites -1 and -2 are commonly occupied by the product and substrates, suggesting that equivocal recognition occurs at the other subsites, which contributes to the wide substrate specificity of this enzyme.
- Davies C, Muirhead H
- Structure of native phosphoglucose isomerase from rabbit: conformational changes associated with catalytic function.
- Acta Crystallogr D Biol Crystallogr. 2003; 59: 453-65
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Phosphoglucose isomerase (PGI) is a housekeeping enzyme of metabolism that catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, with roles in the glycolytic and gluconeogenic pathways. PGI is also a multifunctional protein that manifests the properties of a cytokine in a wide array of cellular processes, including the production of immunoglobulin by B cells and tumour-cell differentiation. The crystal structure of PGI in the native form from rabbit muscle has been solved at a resolution of 2.5 A by a combination of multiple isomorphous replacement and multi-crystal averaging techniques. Comparison with published structures of rabbit PGI in complex with three inhibitors and with the substrate fructose 6-phosphate reveals a number of conformational changes that may be associated with catalytic function. These occur in the small domain around the sugar phosphate-binding site, in a small helix carrying His388 and in a helix near the C-terminal end. One of these may be the structural rearrangement that has been postulated to be the rate-limiting step for catalysis.
- Mayans O, Ivens A, Nissen LJ, Kirschner K, Wilmanns M
- Structural analysis of two enzymes catalysing reverse metabolic reactions implies common ancestry.
- EMBO J. 2002; 21: 3245-54
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The crystal structure of the dimeric anthranilate phosphoribosyltransferase (AnPRT) reveals a new category of phosphoribosyltransferases, designated as class III. The active site of this enzyme is located within the flexible hinge region of its two-domain structure. The pyrophosphate moiety of phosphoribosylpyrophosphate is co-ordinated by a metal ion and is bound by two conserved loop regions within this hinge region. With the structure of AnPRT available, structural analysis of all enzymatic activities of the tryptophan biosynthesis pathway is complete, thereby connecting the evolution of its enzyme members to the general development of metabolic processes. Its structure reveals it to have the same fold, topology, active site location and type of association as class II nucleoside phosphorylases. At the level of sequences, this relationship is mirrored by 13 structurally invariant residues common to both enzyme families. Taken together, these data imply common ancestry of enzymes catalysing reverse biological processes--the ribosylation and deribosylation of metabolic pathway intermediates. These relationships establish new links for enzymes involved in nucleotide and amino acid metabolism.
- Morais MC, Zhang W, Baker AS, Zhang G, Dunaway-Mariano D, Allen KN
- The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily.
- Biochemistry. 2000; 39: 10385-96
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Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.
- Radfar R et al.
- The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica.
- Biochemistry. 2000; 39: 3920-6
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The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.
- Svensson S, Hoog JO, Schneider G, Sandalova T
- Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency.
- J Mol Biol. 2000; 302: 441-53
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The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme.
- Jedrzejas MJ, Chander M, Setlow P, Krishnasamy G
- Mechanism of catalysis of the cofactor-independent phosphoglycerate mutase from Bacillus stearothermophilus. Crystal structure of the complex with 2-phosphoglycerate.
- J Biol Chem. 2000; 275: 23146-53
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The structure of the complex between the 2, 3-diphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus and its 3-phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-phosphoglycerate and two Mn(2+) ions at 1. 7-A resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2- or 3-phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase.
- van den Ent F, Lockhart A, Kendrick-Jones J, Lowe J
- Crystal structure of the N-terminal domain of MukB: a protein involved in chromosome partitioning.
- Structure. 1999; 7: 1181-7
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BACKGROUND: The 170 kDa protein MukB has been implicated in ATP-dependent chromosome partitioning during cell division in Escherichia coli. MukB shares its dimeric structure and domain architecture with the ubiquitous family of SMC (structural maintenance of chromosomes) proteins that facilitate similar functions. The N-terminal domain of MukB carries a putative Walker A nucleotide-binding region and the C-terminal domain has been shown to bind to DNA. Mutant phenotypes and a domain arrangement similar to motor proteins that move on microtubules led to the suggestion that MukB might be a motor protein acting on DNA. RESULTS: We have cloned, overexpressed and crystallized a 26 kDa protein consisting of 227 N-terminal residues of MukB from E. coli. The structure has been solved using multiple anomalous dispersion and has been refined to 2.2 A resolution. The N-terminal domain of MukB has a mixed alpha/beta fold with a central six-stranded antiparallel beta sheet. The putative nucleotide-binding loop, which is part of an unexpected helix-loop-helix motif, is exposed on the surface and no nucleotide-binding pocket could be detected. CONCLUSIONS: The N-terminal domain of MukB has no similarity to the kinesin family of motor proteins or to any other nucleotide-binding protein. Together with the finding of the exposed Walker A motif this observation supports a model in which the N- and C-terminal domains come together in the dimer of MukB to form the active site. Conserved residues on one side of the molecule delineate a region of the N-terminal domain that is likely to interact with the C-terminal domain.
- Thompson TB, Thomas MG, Escalante-Semerena JC, Rayment I
- Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site.
- Biochemistry. 1999; 38: 12995-3005
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The X-ray crystal structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium bound to GMP has been determined by molecular replacement to 2.2 A resolution. CobU is a bifunctional enzyme, which catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring and subsequently functions as a guanylyltransferase to form adenosylcobinamide.GDP. The transferase activity involves a covalent enzyme-guanylyl intermediate that is most likely a phosphoramidate linkage to His(46). Previous studies have shown that the enzyme is a homotrimer and adopts a pinwheel shape. Each subunit consists of a single domain of six parallel beta-strands and one antiparallel strand flanked on either side by a total of five alpha-helices and one helical turn. Interestingly, His(46) in the apoenzyme is located a considerable distance from the kinase active site or P-loop motif and is solvent-exposed [Thompson, T. B., et al. (1998) Biochemistry 37, 7686-7695]. To examine the structural relationship of the two active sites, CobU was cocrystallized with GTP and pyrophosphate. Crystals belong to space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 58. 4 A, b = 87.8 A, and c = 101.6 A. The structure shows electron density for the hydrolysis product GMP rather than the expected covalent guanylyl intermediate which appears to have been hydrolyzed in the crystal lattice. Even so, CobU exhibits a substantial conformational rearrangement. The helix axis containing His(46), the site of guanylylation, rotates 30 degrees and translates 11 A relative to the apo structure and is accompanied by compensatory unwinding and rewinding at the helix ends to allow the induction of a guanosine binding pocket between beta-strand 2 and alpha-helix 2. This conformational change brings the C(alpha) of His(46) approximately 10 A closer to the P-loop motif such that a phosphate ion located in the P-loop is only 6 A from the alpha-phosphate of GMP. This suggests that the P-loop motif may be used to coordinate the terminal phosphates in both the transferase and kinase reactions and implies that the active sites for both reactions overlap.
- van Asselt EJ, Dijkstra AJ, Kalk KH, Takacs B, Keck W, Dijkstra BW
- Crystal structure of Escherichia coli lytic transglycosylase Slt35 reveals a lysozyme-like catalytic domain with an EF-hand.
- Structure. 1999; 7: 1167-80
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BACKGROUND: Lytic transglycosylases are bacterial muramidases that catalyse the cleavage of the beta- 1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydrobond in the MurNAc residue. These muramidases play an important role in the metabolism of the bacterial cell wall and might therefore be potential targets for the rational design of antibacterial drugs. One of the lytic transglycosylases is Slt35, a naturally occurring soluble fragment of the outer membrane bound lytic transglycosylase B (MltB) from Escherichia coli. RESULTS: The crystal structure of Slt35 has been determined at 1.7 A resolution. The structure reveals an ellipsoid molecule with three domains called the alpha, beta and core domains. The core domain is sandwiched between the alpha and beta domains. Its fold resembles that of lysozyme, but it contains a single metal ion binding site in a helix-loop-helix module that is surprisingly similar to the eukaryotic EF-hand calcium-binding fold. Interestingly, the Slt35 EF-hand loop consists of 15 residues instead of the usual 12 residues. The only other prokaryotic proteins with an EF-hand motif identified so far are the D-galactose-binding proteins. Residues from the alpha and core domains form a deep groove where the substrate fragment GlcNAc can be bound. CONCLUSIONS: The three-domain structure of Slt35 is completely different from the Slt70 structure, the only other lytic transglycosylase of known structure. Nevertheless, the core domain of Slt35 closely resembles the fold of the catalytic domain of Slt70, despite the absence of any obvious sequence similarity. Residue Glu162 of Slt35 is in an equivalent position to Glu478, the catalytic acid/base of Slt70. GlcNAc binds close to Glu162 in the deep groove. Moreover, mutation of Glu162 into a glutamine residue yielded a completely inactive enzyme. These observations indicate the location of the active site and strongly support a catalytic role for Glu162.
- Van Asselt EJ, Perrakis A, Kalk KH, Lamzin VS, Dijkstra BW
- Accelerated X-ray structure elucidation of a 36 kDa muramidase/transglycosylase using wARP.
- Acta Crystallogr D Biol Crystallogr. 1998; 54: 58-73
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The X-ray structure of the 36 kDa soluble lytic transglycosylase from Escherichia coli has been determined starting with the multiple isomorphous replacement method with inclusion of anomalous scattering at 2.7 A resolution. Subsequently, before any model building was carried out, phases were extended to 1.7 A resolution with the weighted automated refinement procedure wARP, which gave a dramatic improvement in the phases. The electron-density maps from wARP were of outstanding quality for both the main chain and the side chains of the protein, which allowed the time spent on the tracing, interpretation and building of the X-ray structure to be substantially shortened. The structure of the soluble lytic transglycosylase was refined at 1.7 A resolution with X-PLOR to a final crystallographic R factor of 18.9%. Analysis of the wARP procedure revealed that the use of the maximum-likelihood refinement in wARP gave much better phases than least-squares refinement, provided that the ratio of reflections to protein atom parameters was approximately 1.8 or higher. Furthermore, setting aside 5% of the data for an Rfree test set had a negative effect on the phase improvement. The mean WwARP, a weight determined at the end of the wARP procedure and based on the variance of structure factors from six individually refined wARP models, proved to be a better indicator than the Rfree factor to judge different phase improvement protocols. The elongated Slt35 structure has three domains named the alpha, beta and core domains. The alpha domain contains mainly alpha-helices, while the beta domain consists of a five-stranded antiparallel beta-sheet flanked by a short alpha-helix. Sandwiched between the alpha and beta domains is the core domain, which bears some resemblance to the fold of the catalytic domain of the previously elucidated 70 kDa soluble lytic transglycosylase from E. coli. The putative active site is at the bottom of a large deep groove in the core domain.
- Campobasso N, Costello CA, Kinsland C, Begley TP, Ealick SE
- Crystal structure of thiaminase-I from Bacillus thiaminolyticus at 2.0 A resolution.
- Biochemistry. 1998; 37: 15981-9
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Thiaminase-I catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles, such as pyridine, aniline, catechols, quinoline, and cysteine. The crystal structure of the enzyme from Bacillus thiaminolyticus was determined at 2.5 A resolution by multiple isomorphous replacement and refined to an R factor of 0.195 (Rfree = 0.272). Two other structures, one native and one containing a covalently bound inhibitor, were determined at 2.0 A resolution by molecular replacement from a second crystal form and were refined to R factors of 0.205 and 0.217 (Rfree = 0.255 and 0.263), respectively. The overall structure contains two alpha/beta-type domains separated by a large cleft. At the base of the cleft lies Cys113, previously identified as a key active site nucleophile. The structure with a covalently bound thiamin analogue, which functions as a mechanism-based inactivating agent, confirms the location of the active site. Glu241 appears to function as an active site base to increase the nucleophilicity of Cys113. The mutant Glu241Gln was made and shows no activity. Thiaminase-I shows no sequence identity to other proteins in the sequence databases, but the three-dimensional structure shows very high structural homology to the periplasmic binding proteins and the transferrins.
- Saxild HH, Andersen LN, Hammer K
- Dra-nupC-pdp operon of Bacillus subtilis: nucleotide sequence, induction by deoxyribonucleosides, and transcriptional regulation by the deoR-encoded DeoR repressor protein.
- J Bacteriol. 1996; 178: 424-34
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The genes encoding deoxyriboaldolase (dra), nucleoside uptake protein (nupC), and pyrimidine nucleoside sequences were determined. Sequence analysis showed that the genes were localized immediately downstream of the hut operon. Insertional gene disruption studies indicated that the three genes constitute an operon with the gene order dra-nupC-pdp. A promoter mapping immediately upstream of the dra gene was identified, and downstream of the pdp gene the nucleotide sequence indicated the existence of a factor-independent transcription terminator structure. In wild-type cells growing in succinate minimal medium, the pyrimidine nucleoside phosphorylase and deoxyriboaldolase levels were five- to eightfold higher in the presence of thymidine and fourfold higher in the presence of deoxyadenosine. By the use of lacZ fusions, the regulation was found to be at the level of transcription. The operon expression was subject to glucose repression. Upstream of the dra gene an open reading frame of 313 amino acids was identified. Inactivation of this gene led to an approximately 10-fold increase in the levels of deoxyriboaldolase and pyrimidine nucleoside phosphorylase, and no further induction was seen upon the addition of deoxyribonucleosides. The upstream gene most likely encodes the regulator for the dra-nupC-pdp operon and was designated deoR (stands for deoxyribonucleoside regulator).
- Qiu X, Pohl E, Holmes RK, Hol WG
- High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor.
- Biochemistry. 1996; 35: 12292-302
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The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 A resolution and in complex with Mn2+ at 2.2 A resolution. The structure of the flexible third domain could be determined at this high resolution. It appears to contain five antiparallel strands exhibiting a fold very similar to the SH3 domain. A superposition of 46 equivalent C alpha atoms of DtxR and alpha-spectrin SH3 resulted in an rms deviation of 3.0 A. The sequence identity is only 7%. This third domain of DtxR appears to have no interactions with the DNA binding domain nor with the metal binding domain of the repressor. Yet, flexibility in the region between the second and the third domain allows in principle significant conformational changes such as might occur upon DNA binding. The two metal binding sites in the second domain have been unraveled in considerable detail. Metal binding site 1 was well occupied in both the cobalt and manganese structures and showed a surprising sulfate ion as ligand. The sulfate was proven beyond doubt by the high peak at its position in a selenate versus sulfate difference Fourier. The presence of the intriguing sulfate ion at such a crucial position near the metal corepressor suggests the possibility that under physiological conditions phosphate may act as a "co-corepressor" for this class of metal-regulated DNA binding proteins in Corynebacteria, Mycobacteria, and related organisms. The second metal binding site is significantly different in these two DtxR structures. In the 2.0 A cobalt structure, the site is not occupied by a metal ion. In the 2.2 A manganese structure the site is well occupied, at approximately the same position as observed previously in cadmium DtxR. The ligands are Glu105, His106, the carbonyl oxygen of Cys102, and a water molecule. The reasons for differential occupancy of this site in different structures are intriguing and require further investigations.
- Mattevi A, Valentini G, Rizzi M, Speranza ML, Bolognesi M, Coda A
- Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition.
- Structure. 1995; 3: 729-41
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BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation. RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme. CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.
- Cooper JB et al.
- X-ray structure analysis of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis at 2.0 Angstroms resolution reveals novel dimer-dimer interactions.
- J Mol Biol. 1995; 246: 531-44
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The X-ray structure of the tetrameric iron-dependent superoxide dismutase from Mycobacterium tuberculosis has been refined to an R-factor of 0.167 and a correlation coefficient of 0.954 at 2.0 A resolution. The crystals are monoclinic P2(1) and have four subunits related by strong non-crystallographic 222 (or D2) symmetry in the asymmetric unit. 198 of the 207 amino acids of each subunit are defined by the electron density which shows that they adopt the conserved fold of other iron- or manganese-dependent SODs. The structure can be divided into two domains, the N-terminal domain involving an extended region followed by two projecting antiparallel alpha-helices, and the C-terminal domain containing four more helical segments with a three-stranded antiparallel beta-sheet inserted sequentially between the fourth and fifth helices. The catalytic iron is co-ordinated by five ligands: three histidines (residues 28, 76 and 164), one aspartate (160) and a solvent molecule. The inferred positions of protons at the active site are consistent with the solvent ligand being a hydroxide ion. This ligand interacts with His145 in the Mycobacterium tuberculosis SOD. In the highly homologous Mycobacterium leprae Mn-SOD, the histidine is replaced by glutamine, this being the only significant residue difference within 10 A of the Fe3+. The nature of the amino acid at this position may influence the metal ion specificity of these enzymes. The subunits of the Mycobacterium tuberculosis SOD associate by polar contacts to form dimers, which closely resemble those of other dimeric or tetrameric Fe- or Mn-SODs. However, the dimer-dimer interactions within the tetramer are novel, being dominated by dimerisation of the 144 to 152 loop regions which connect the outer two beta-strands of the three-membered beta-sheet. This contrasts strongly with the other tetrameric Fe- or Mn-SODs where the dimer-dimer association is dominated by the projecting alpha alpha-turn in the N-terminal domain.
- Machius M, Wiegand G, Huber R
- Crystal structure of calcium-depleted Bacillus licheniformis alpha-amylase at 2.2 A resolution.
- J Mol Biol. 1995; 246: 545-59
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The three-dimensional structure of the calcium-free form of Bacillus licheniformis alpha-amylase (BLA) has been determined by multiple isomorphous replacement in a crystal of space group P4(3)2(1)2 (a = b = 119.6 A, c = 85.4 A). The structure was refined using restrained crystallographic refinement to an R-factor of 0.177 for 28,147 independent reflections with intensities FObs > 0 at 2.2 A resolution, with root mean square deviations of 0.008 A and 1.4 degrees from ideal bond lengths and bond angles, respectively. The final model contains 469 residue, 237 water molecules, and one chloride ion. The segment between Trp182 and Asn192 could not be located in the electron density, nor could the N and C termini. Cleavage of the calcium-free form of BLA was observed after Glu189, due to a Glu-C endopeptidase present in trace amounts in the preparation. BLA did not crystallize without this cleavage under the conditions applied. BLA exhibits the characteristic overall topological fold observed for other alpha-amylases and related amylolytic enzymes: a central domain A containing an alpha/beta-barrel with a large protrusion between beta-strand 3 and alpha-helix 3 (domain B) and a C-terminal greek key motif (domain C). Unlike in the other enzymes, domain B possesses a beta-sheet made up of six loosely connected, twisted beta-strands forming a kind of a barrel with a large hole in the interior. Topological comparisons to TAKA-amylase, pig pancreatic alpha-amylase and cyclodextrin glycosyltransferase reveal a very high structural equivalence for large portions of the proteins and an exceptionally pronounced structural similarity for calcium binding, chloride binding and the active site. None of the theories proposed to explain the enhanced thermostability of BLA showed a satisfactory correlation with the three-dimensional structure. Instead, sequence comparisons to the less thermostable bacterial alpha-amylase from Bacillus amyloliquefaciens (BAA) indicate that some ionic interactions present in BLA, but which cannot be formed in BAA, might be responsible for the enhanced thermostability of BLA.
- Moghaddam A et al.
- Thymidine phosphorylase is angiogenic and promotes tumor growth.
- Proc Natl Acad Sci U S A. 1995; 92: 998-1002
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Platelet-derived endothelial cell growth factor was previously identified as the sole angiogenic activity present in platelets; it is now known to be thymidine phosphorylase (TP). The effect of TP on [methyl-3H]thymidine uptake does not arise from de novo DNA synthesis and the molecule is not a growth factor. Despite this, TP is strongly angiogenic in a rat sponge and freeze-injured skin graft model. Neutralizing antibodies and site-directed mutagenesis confirmed that the enzyme activity of TP is a condition for its angiogenic activity. The level of TP was found to be elevated in human breast tumors compared to normal breast tissue (P < 0.001). Overexpression of TP in MCF-7 breast carcinoma cells had no effect on growth in vitro but markedly enhanced tumor growth in vivo. These data and the correlation of expression in tumors with malignancy identify TP as a target for antitumor strategies.
- Mirwaldt C, Korndorfer I, Huber R
- The crystal structure of dihydrodipicolinate synthase from Escherichia coli at 2.5 A resolution.
- J Mol Biol. 1995; 246: 227-39
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The crystal structure of dihydrodipicolinate synthase from E. coli was determined by multiple isomorphous replacement methods. The structure was refined at a resolution of 2.5 A and the final R-factor is 19.6% for 32,190 reflections between 10.0 A and 2.5 A and F > 2 sigma (F). The crystallographic asymmetric unit contains two monomers related by approximate 2-fold symmetry. A tetramer with approximate 222 symmetry is built up by crystallographic symmetry. The tetramer is almost planar with no contacts between the subunits related by the non-crystallographic dyad. The active sites are accessible from a wide water-filled channel in the center of the tetramer. The dihydrodipicolinate synthase monomer is composed of two domains. Each polypeptide chain is folded into an 8-fold alpha/beta barrel and a C-terminal alpha-helical domain comprising residues 224 to 292. The fold is similar to that of N-acetylneuraminate lyase. The active site lysine 161 is located in the alpha/beta barrel and has access via two entrances from the C-terminal side of the barrel.
- Wolodko WT, Fraser ME, James MN, Bridger WA
- The crystal structure of succinyl-CoA synthetase from Escherichia coli at 2.5-A resolution.
- J Biol Chem. 1994; 269: 10883-90
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The x-ray crystal structure of succinyl-CoA synthetase (SCS) from Escherichia coli has been determined by the method of multiple isomorphous replacement to a resolution of 2.5 A. Crystals of SCS are tetragonal with a space group of P4(3)22 and unit cell dimensions of a = b = 98.47 A and c = 400.6 A. One molecule of SCS (142 kDa) is contained in the asymmetric unit. The current model has been refined to a conventional R factor of 21.6% with root mean square deviations from ideal stereochemistry of 0.022 A for bond lengths and 3.25 degrees for bond angles. The quaternary organization of the E. coli enzyme is an alpha 2 beta 2 heterotetramer. In this tetramer, the alpha-subunits interact only with the beta-subunits, whereas the beta-subunits interact to form the dimer of alpha beta-dimers. The two active site pockets are located at regions of contact between alpha- and beta-subunits. One molecule of coenzyme A is bound to each alpha-subunit at a typical nucleotide-binding motif, and His-246 of each alpha-subunit is phosphorylated. This phosphohistidine, a catalytic intermediate, is stabilized by two helix dipoles (the "power" helices), one from each of the two subunit types. A short segment of the beta-subunit from one alpha beta-dimer is in close proximity to the CoA-binding site of the other alpha beta-dimer, providing a possible rationale for the overall tetrameric structure.
- Yamaguchi H et al.
- Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution.
- J Mol Biol. 1993; 229: 1083-100
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Glutathione synthetase (gamma-L-glutamyl-L-cysteine: glycine ligase (ADP-forming) EC 18.104.22.168: GSHase) catalyzes the synthesis of glutathione from gamma-L-glutamyl-L-cysteine and Gly in the presence of ATP. The enzyme from Escherichia coli is a tetramer with four identical subunits of 316 amino acid residues. The crystal structure of the enzyme has been determined by isomorphous replacement and refined to a 2.0 A resolution. Two regions, Gly164 to Gly167 and Ile226 to Arg241, are invisible on the electron density map. The refined model of the subunit includes 296 amino acid residues and 107 solvent molecules. The crystallographic R-factor is 18.6% for 17.914 reflections with F > 3 sigma between 6.0 A and 2.0 A. The structure consists of three domains: the N-terminal, central, and C-terminal domains. In the tetrameric molecule, two subunits that are in close contact form a tight dimer, two tight dimers forming a tetramer with two solvent regions. The ATP molecule is located in the cleft between the central and C-terminal domains. The ATP binding site is surrounded by two sets of the structural motif that belong to those respective domains. Each motif consists of an anti-parallel beta-sheet and a glycine-rich loop.
- Jelsch C, Lenfant F, Masson JM, Samama JP
- Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution.
- FEBS Lett. 1992; 299: 135-42
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The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.
- Worthylake D, Meadow ND, Roseman S, Liao DI, Herzberg O, Remington SJ
- Three-dimensional structure of the Escherichia coli phosphocarrier protein IIIglc.
- Proc Natl Acad Sci U S A. 1991; 88: 10382-6
- Display abstract
The crystal structure of a proteolytically modified form of the Escherichia coli phosphocarrier and signal transducing protein IIIglc has been determined by multiple isomorphous and molecular replacement. The model has been refined to an R-factor of 0.166 for data between 6- and 2.1-A resolution with an rms deviation of 0.020 A from ideal bond lengths and 3.2 degrees from ideal bond angles. The molecule is a beta-sheet sandwich, with six antiparallel strands on either side. Several short distorted helices line the periphery of the active site, which is a shallow extremely hydrophobic depression approximately 18 A in diameter near the center of one face. The side chains of the active site histidine residues 75 and 90 face each other at the center of the depression, with the N3 positions exposed to solvent, separated by 3.3 A in an excellent position to form adducts with phosphate. Chloroplatinate forms a divalent adduct with both histidyl side chains, suggesting that the phosphodonor reaction might proceed through a similar transition state. The hydrophobic patch forms the primary crystal contact, suggesting a mode of association of IIIglc with other components of the phosphoenolpyruvate-dependent phosphotransferase system.
- Vrielink A, Lloyd LF, Blow DM
- Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution.
- J Mol Biol. 1991; 219: 533-54
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Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 22.214.171.124) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.
- Katayanagi K et al.
- Three-dimensional structure of ribonuclease H from E. coli.
- Nature. 1990; 347: 306-9
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The three-dimensional structure of RNase H from Escherichia coli was determined at 1.8 A resolution by X-ray crystallography. The enzyme was found to belong to the alpha + beta class of structures, consisting of two distinct domains. The structure implies a possible region interacting with a DNA-RNA hybrid. The Mg2(+)-binding site essential for activity is located near a cluster of four acidic amino acids--one glutamic and three aspartic acid residues. These residues are completely conserved in the homology alignment of sequences of RNase H and reverse transcriptases from retroviruses and retrovirus-like entities. The structural motif of beta strands around the Mg2(+)-binding site has similarities to that in DNase I.
- Vellieux FM et al.
- Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.
- EMBO J. 1989; 8: 2171-8
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The three-dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X-ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc-shaped domain. This domain is formed by a circular arrangement of seven topologically identical four-stranded antiparallel beta-sheets, with approximately 7-fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta-sheet scaffold of a central three-stranded antiparallel sheet flanked by two shorter two-stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co-factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co-factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.
- Hoeffken HW, Knof SH, Bartlett PA, Huber R, Moellering H, Schumacher G
- Crystal structure determination, refinement and molecular model of creatine amidinohydrolase from Pseudomonas putida.
- J Mol Biol. 1988; 204: 417-33
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The three-dimensional crystal structure of creatine amidinohydrolase (creatinase EC 126.96.36.199) from Pseudomonas putida, a dimeric enzyme with a molecular weight of 97,000, has been determined by multiple isomorphous replacement, averaging over the local dyad and restrained crystallographic refinement at 1.9 A with a crystallographic R-value of 17.7%. The asymmetric unit contains a dimer. The two chemically identical subunits consist of 403 residues each. A subunit is built up of two domains, a small N-terminal and a larger C-terminal domain. The small domain has a central seven-stranded beta pleated sheet with short helices on the outside. The large domain forms a six-stranded antiparallel beta half-barrel with helices on the outside. The two domains are connected by a segment that links two helices. The binding site of the competitive inhibitor carbamoyl sarcosine, a close analog of the substrate creatine, is located in the center of the large domain and partly covered by the small domain of the other subunit. The carbamoyl group is tightly co-ordinated to a water molecule, which presumably represents the nucleophile involved in hydrolysis of creatine. A catalytic mechanism is proposed on the basis of this structure.
- el Kouni MH, Naguib FN, Niedzwicki JG, Iltzsch MH, Cha S
- Uridine phosphorylase from Schistosoma mansoni.
- J Biol Chem. 1988; 263: 6081-6
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Uridine phosphorylase is the only pyrimidine nucleoside cleaving activity that can be detected in extracts of Schistosoma mansoni. The enzyme is distinct from the two purine nucleoside phosphorylases contained in this parasite. Although Urd is the preferred substrate, uridine phosphorylase can also catalyze the reversible phosphorolysis of dUrd and dThd, but not Cyd, dCyd, or orotidine. The enzyme was purified 170-fold to a specific activity of 2.76 nmol/min/mg of protein with a 16% yield. It has a Mr of 56,000 as determined by molecular sieving on Sephadex G-100. The mechanism of uridine phosphorylase is sequential. When Urd was the substrate, the KUrd = 13 microM and the KPi = 533 +/- 78 microM. When dThd was used as a substrate, the KdThd = 54 microM and the KPi = 762 +/- 297 microM. The Vmax with dThd was 53 +/- 9.8% that of Urd. dThd was a competitive inhibitor when Urd was used as a substrate. The enzyme showed substrate inhibition by Urd, dThd (greater than 0.125 mM) and phosphate (greater than 10 mM). 5-(Benzyloxybenzyloxybenzyl)acyclouridine was identified as a potent and specific inhibitor of parasite (Ki = 0.98 microM) but not host uridine phosphorylase. Structure-activity relationship studies suggest that uridine phosphorylase from S. mansoni has a hydrophobic pocket adjacent to the 5-position of the pyrimidine ring and indicate differences between the binding sites of the mammalian and parasite enzymes. These differences may be useful in designing specific inhibitors for schistosomal uridine phosphorylase which will interfere selectively with nucleic acids synthesis in this parasite.
- Desgranges C, Razaka G, Rabaud M, Bricaud H
- Catabolism of thymidine in human blood platelets: purification and properties of thymidine phosphorylase.
- Biochim Biophys Acta. 1981; 654: 211-8
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A pyrimidine nucleoside phosphorylase was partially purified from human blood platelets. The purified enzyme, as well as crude enzyme preparations, catalyses the phosphorolysis of thymidine and deoxyuridine, but not of uridine, and is able to catalyse direct pentosyl transfer from these deoxyribonucleosides to uracil or thymine; this enzyme has the properties of a thymidine phosphorylase. It has a molecular weight of about 110,000 and is composed of two identical subunits; it is phosphate dependent, has a maximal activity at a pH value of 5.7, and an isoelectric point of 4.4. This enzyme was mainly of cytoplasmic origin. Although platelet thymidine phosphorylase could promote the degradation or synthesis of thymidine, intact platelets degraded thymidine but were not able to synthesize thymidine from thymine. Blood platelets may play an important role in the degradation of plasma thymidine.
- Hoffee PA, Blank J
- Thymidine phosphorylase from Salmonella typhimurium.
- Methods Enzymol. 1978; 51: 437-42
- Sprang S, Fletterick RJ
- Crystallographic analysis of phosphorylase alpha at 2.5 A resolution, a comment on the chemical sequence.
- Biochemistry. 1978; 17: 5693-4
- Evans DR et al.
- Aqueous central cavity in aspartate transcarbamylase from Escherichia coli.
- Science. 1973; 179: 683-5
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A three-dimensional x-ray diffraction study of aspartate transcarbamylase to 5.5-angstrom resolution, with the aid of four isomorphous heavy atom derivatives, indicates the presence of a central aqueous cavity approximating an oblate spheroid about 25 by 50 by 50 angstroms in dimension, within a molecule about 90 by 110 by 110 angstroms in largest dimensions.