NORMAL GENOMIC

• Brito-Arias M, Aguilar-Lemus C, Hurtado-Ponce PB, Martinez-Barron G, Ibanez-Hernandez M
• Synthesis of phenylazonaphtol-beta-D-O-glycosides, evaluation as substrates for beta-glycosidase activity and molecular studies.
• Org Med Chem Lett. 2014; 4: 2-2
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• BACKGROUND: Phenylazonaphtol-beta-D-O-glycosides are alternative substrates for the detection of enzymatic activity of beta-glycosidases which are involved in various important processes. These azoic compounds are currently exploited as prodrugs for colonic disease due the presence of beta-glycosidase activity in the gut flora and therefore allowing the release of the drug at the specific site. RESULTS: Phenylazonaphtol-beta-D-O-glucoside 3a and galactoside 3b were prepared via diazonium salt conditions under weak acidic conditions which do not compromise the O-glycosidic bond stability, by coupling reaction between 2-naphtol sodium salt with aminoglycosides 1a and 1b. The resulting phenylazonaphtol glycosides 2a and 2b were deprotected affording the phenylazonaphtol glycosides 3a and 3b in quantitative yield. The galactoside glycoside 3b was assayed as substrate for in vitro beta-galactosidase enzymatic activity showing strong absorbance after releasing of the azoic chromophore. Also, docking studies were performed to determine the best pose as well as the interactions between the ligand and the residues located at the active site. CONCLUSIONS: The methodology developed for synthesizing the phenylazonaphtol glycosides described proved to be convenient for generating azoic functionalities in the presence of glycosidic bonds and the glycosides suitable as alternative substrates and potentially useful prodrugs in the treatment of colonic diseases.

• Lee EG, Kim S, Oh DB, Lee SY, Kwon O
• Distinct roles of beta-galactosidase paralogues of the rumen bacterium Mannheimia succiniciproducens.
• J Bacteriol. 2012; 194: 426-36
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• Mannheimia succiniciproducens, a rumen bacterium belonging to the family Pasteurellaceae, has two putative beta-galactosidase genes, bgaA and bgaB, encoding polypeptides whose deduced amino acid sequences share 56% identity with each other and show approximately 30% identity to the Escherichia coli gene for LacZ. The M. succiniciproducens bgaA (MsbgaA) gene-deletion mutant was not able to grow on lactose as the sole carbon source, suggesting its essential role in lactose metabolism, whereas the MsbgaB gene-deletion mutant did not show any growth defect on a lactose medium. Furthermore, the expression of the MsbgaA gene was induced by the addition of lactose in the growth medium, whereas the MsbgaB gene was constitutively expressed independently of a carbon source. Biochemical characterization of the recombinant proteins revealed that MsBgaA is more efficient than MsBgaB in hydrolyzing o-nitrophenyl-beta-d-galactopyranoside and p-nitrophenyl-beta-d-galactopyranoside. MsBgaA was highly specific for the hydrolysis of lactose, with a catalytic efficiency of 46.9 s(-1) mM(-1). However, MsBgaB was more efficient for the hydrolysis of lactulose than lactose, and the catalytic efficiency was 10.0 s(-1) mM(-1). Taken together, our results suggest that the beta-galactosidase paralogues of M. succiniciproducens BgaA and BgaB play a critical role in lactose metabolism and in an unknown but likely specific function for rumen bacteria, respectively.

• Zhao F et al.
• beta-Galactosidase-instructed formation of molecular nanofibers and a hydrogel.
• Nanoscale. 2011; 3: 2859-61
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• Here we report the first example of using beta-galactosidase to trigger the formation of cell compatible, supramolecular nanofibers, which ultimately may lead to a new approach for the development of soft nanotechnology.

• Gloster TM, Davies GJ
• Glycosidase inhibition: assessing mimicry of the transition state.
• Org Biomol Chem. 2010; 8: 305-20
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• Glycoside hydrolases, the enzymes responsible for hydrolysis of the glycosidic bond in di-, oligo- and polysaccharides, and glycoconjugates, are ubiquitous in Nature and fundamental to existence. The extreme stability of the glycosidic bond has meant these enzymes have evolved into highly proficient catalysts, with an estimated 10(17) fold rate enhancement over the uncatalysed reaction. Such rate enhancements mean that enzymes bind the substrate at the transition state with extraordinary affinity; the dissociation constant for the transition state is predicted to be 10(-22) M. Inhibition of glycoside hydrolases has widespread application in the treatment of viral infections, such as influenza and HIV, lysosomal storage disorders, cancer and diabetes. If inhibitors are designed to mimic the transition state, it should be possible to harness some of the transition state affinity, resulting in highly potent and specific drugs. Here we examine a number of glycosidase inhibitors which have been developed over the past half century, either by Nature or synthetically by man. A number of criteria have been proposed to ascertain which of these inhibitors are true transition state mimics, but these features have only be critically investigated in a very few cases.

• Wlodawer A, Minor W, Dauter Z, Jaskolski M
• Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures.
• FEBS J. 2008; 275: 1-21
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• The number of macromolecular structures deposited in the Protein Data Bank now exceeds 45,000, with the vast majority determined using crystallographic methods. Thousands of studies describing such structures have been published in the scientific literature, and 14 Nobel prizes in chemistry or medicine have been awarded to protein crystallographers. As important as these structures are for understanding the processes that take place in living organisms and also for practical applications such as drug design, many non-crystallographers still have problems with critical evaluation of the structural literature data. This review attempts to provide a brief outline of technical aspects of crystallography and to explain the meaning of some parameters that should be evaluated by users of macromolecular structures in order to interpret, but not over-interpret, the information present in the coordinate files and in their description. A discussion of the extent of the information that can be gleaned from the coordinates of structures solved at different resolution, as well as problems and pitfalls encountered in structure determination and interpretation are also covered.

• Addlagatta A, Gay L, Matthews BW
• Structural basis for the unusual specificity of Escherichia coli aminopeptidase N.
• Biochemistry. 2008; 47: 5303-11
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• Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.

• 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.

• Xiong AS et al.
• Directed evolution of beta-galactosidase from Escherichia coli into beta-glucuronidase.
• J Biochem Mol Biol. 2007; 40: 419-25
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• In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. E. coli beta-galactosidase and beta-glucuronidase are widely used, and their biological function, catalytic mechanism, and molecular structures are well characterized. We applied an in vitro directed evolution strategy through DNA shuffling and obtained five mutants named YG6764, YG6768, YG6769, YG6770 and YG6771 after two rounds of DNA shuffling and screening, which exhibited more beta-glucuronidase activity than wild-type beta-galactosidase. These variants had mutations at fourteen nucleic acid sites, resulting in changes in ten amino acids: S193N, T266A, Q267R, V411A, D448G, G466A, L527I, M543I, Q626R and Q951R. We expressed and purified those mutant proteins. Compared to the wild-type protein, five mutant proteins exhibited high beta-glucuronidase activity. The comparison of molecular models of the mutated and wildtype enzymes revealed the relationship between protein function and structural modification.

• 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.

• Arjunan P et al.
• A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.
• J Biol Chem. 2006; 281: 15296-303
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• The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

• Graham SC et al.
• Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: insights into substrate recognition and the mechanism of catalysis.
• Biochemistry. 2006; 45: 964-75
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• Aminopeptidase P (APPro) is a manganese-dependent enzyme that cleaves the N-terminal amino acid from polypeptides where the second residue is proline. APPro shares a similar fold, substrate specificity, and catalytic mechanism with methionine aminopeptidase and prolidase. To investigate the roles of conserved residues at the active site, seven mutant forms of APPro were characterized kinetically and structurally. Mutation of individual metal ligands selectively abolished binding of either or both Mn(II) atoms at the active site, and none of these metal-ligand mutants had detectable catalytic activity. Mutation of the conserved active site residues His243 and His361 revealed that both are required for catalysis. We propose that His243 stabilizes substrate binding through an interaction with the carbonyl oxygen of the requisite proline residue of a substrate and that His361 stabilizes substrate binding and the gem-diol catalytic intermediate. Sequence, structural, and kinetic analyses reveal that His350, conserved in APPro and prolidase but not in methionine aminopeptidase, forms part of a hydrophobic binding pocket that gives APPro its proline specificity. Further, peptides in which the required proline residue is replaced by N-methylalanine or alanine are cleaved by APPro, but they are extremely poor substrates due to a loss of interactions between the prolidyl ring of the substrate and the hydrophobic proline-binding pocket.

• Spiegel K, De Grado WF, Klein ML
• Structural and dynamical properties of manganese catalase and the synthetic protein DF1 and their implication for reactivity from classical molecular dynamics calculations.
• Proteins. 2006; 65: 317-30
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• There is a pressing need for accurate force fields to assist in metalloprotein analysis and design. Recent work on the design of mimics of dimetal proteins highlights the requirements for activity. DF1 is a de novo designed protein, which mimics the overall fold and active site geometry of a series of diiron and dimanganese proteins. Specifically, the dimanganese form of DF1 is a mimic of the natural enzyme manganese catalase, which catalyzes the dismutation reaction of hydrogen peroxide into water and oxygen. During catalytic turnover, the active site has to accommodate both the reduced and the oxidized state of the dimanganese core. The biomimetic protein DF1 is only stable in the reduced form and thus not active. Furthermore, the synthetic protein features an additional bridging glutamate sidechain, which occupies the substrate binding site. The goal of this study is to develop classical force fields appropriate for design of such important dimanganese proteins. To this aim, we use a nonbonded model to represent the metal-ligand interactions, which implicitly takes into account charge transfer and local polarization effects between the metal and its ligands. To calibrate this approach, we compare and contrast geometric and dynamical properties of manganese catalase and DF1. Having demonstrated a good correspondence with experimental structural data, we examine the effect of mutating the bridging glutamate to aspartate (M1) and serine (M2). Classical MD based on the refined forcefield shows that these point mutations affect not only the immediate coordination sphere of the manganese ions, but also the relative position of the helices, improving the similarity to Mn-catalase, especially in case of M2. On the basis of these findings, classical molecular dynamics calculations with the active site parameterization scheme introduced herein seem to be a promising addition to the protein design toolbox.

• Fujiwara K, Toma S, Okamura-Ikeda K, Motokawa Y, Nakagawa A, Taniguchi H
• Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site.
• J Biol Chem. 2005; 280: 33645-51
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• Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of alpha-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 angstroms resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.

• Fladvad M et al.
• Molecular mapping of functionalities in the solution structure of reduced Grx4, a monothiol glutaredoxin from Escherichia coli.
• J Biol Chem. 2005; 280: 24553-61
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• The ubiquitous glutaredoxin protein family is present in both prokaryotes and eukaryotes, and is closely related to the thioredoxins, which reduce their substrates using a dithiol mechanism as part of the cellular defense against oxidative stress. Recently identified monothiol glutaredoxins, which must use a different functional mechanism, appear to be essential in both Escherichia coli and yeast and are well conserved in higher order genomes. We have employed high resolution NMR to determine the three-dimensional solution structure of a monothiol glutaredoxin, the reduced E. coli Grx4. The Grx4 structure comprises a glutaredoxin-like alpha-beta fold, founded on a limited set of strictly conserved and structurally critical residues. A tight hydrophobic core, together with a stringent set of secondary structure elements, is thus likely to be present in all monothiol glutaredoxins. A set of exposed and conserved residues form a surface region, implied in glutathione binding from a known structure of E. coli Grx3. The absence of glutaredoxin activity in E. coli Grx4 can be understood based on small but significant differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, in contrast with dithiol thioredoxins and glutaredoxins, where differences between oxidized and reduced forms are subtle and local. Structural and functional implications are discussed with particular emphasis on identifying common monothiol glutaredoxin properties in substrate specificity and ligand binding events, linking the thioredoxin and glutaredoxin systems.

• Roderick SL
• The lac operon galactoside acetyltransferase.
• C R Biol. 2005; 328: 568-75
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• Of the proteins encoded by the three structural genes of the lac operon, the galactoside acetyltransferase (thiogalactoside transacetylase, LacA, GAT) encoded by lacA is the only protein whose biological role remains in doubt. Here, we briefly note the classical literature that led to the identification and initial characterization of GAT, and focus on more recent results which have revealed its chemical mechanism of action and its membership in a large superfamily of structurally similar acyltransferases. The structural and sequence similarities of several members of this superfamily confirm the original claim for GAT as a CoA-dependent acetyltransferase specific for the 6-hydroxyl group of certain pyranosides, but do not yet point to the identity of the natural substrate(s) of the enzyme.

• 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.

• Bhaumik P, Koski MK, Bergmann U, Wierenga RK
• Structure determination and refinement at 2.44 A resolution of argininosuccinate lyase from Escherichia coli.
• Acta Crystallogr D Biol Crystallogr. 2004; 60: 1964-70
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• Escherichia coli argininosuccinate lyase has been crystallized from a highly concentrated sample of purified recombinant alpha-methylacyl-CoA racemase, in which it occurred as a minor impurity. The structure has been solved using molecular replacement at 2.44 A resolution. The enzyme is tetrameric, but in this crystal form there is a dimer in the asymmetric unit. The tetramer has four active sites; each active site is constructed from loops of three different subunits. One of these catalytic loops, near residues Ser277 and Ser278, was disordered in previous structures of active lyases, but is very well ordered in this structure in one of the subunits owing to the presence of two phosphate ions in the active-site cavity. The positions of these phosphate ions indicate a plausible mode of binding of the succinate moiety of the substrate in the competent catalytic complex.

• Juers DH, Hakda S, Matthews BW, Huber RE
• Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase.
• Biochemistry. 2003; 42: 13505-11
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• The open-closed conformational switch in the active site of Escherichia coli beta-galactosidase was studied by X-ray crystallography and enzyme kinetics. Replacement of Gly794 by alanine causes the apoenzyme to adopt the closed rather than the open conformation. Binding of the competitive inhibitor isopropyl thio-beta-D-galactoside (IPTG) requires the mutant enzyme to adopt its less favored open conformation, weakening affinity relative to wild type. In contrast, transition-state inhibitors bind to the enzyme in the closed conformation, which is favored for the mutant, and display increased affinity relative to wild type. Changes in affinity suggest that the free energy difference between the closed and open forms is 1-2 kcal/mol. By favoring the closed conformation, the substitution moves the resting state of the enzyme along the reaction coordinate relative to the native enzyme and destabilizes the ground state relative to the first transition state. The result is that the rate constant for galactosylation is increased but degalactosylation is slower. The covalent intermediate may be better stabilized than the second transition state. The substitution also results in better binding of glucose to both the free and the galactosylated enzyme. However, transgalactosylation with glucose to produce allolactose (the inducer of the lac operon) is slower with the mutant than with the native enzyme. This suggests either that the glucose is misaligned for the reaction or that the galactosylated enzyme with glucose bound is stabilized relative to the transition state for transgalactosylation.

• Snyder L, Blight S, Auchtung J
• Regulation of translation of the head protein of T4 bacteriophage by specific binding of EF-Tu to a leader sequence.
• J Mol Biol. 2003; 334: 349-61
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• Recent evidence indicates that translation elongation factor Tu (EF-Tu) has a role in the cell in addition to its well established role in translation. The translation factor binds to a specific region called the Gol region close to the N terminus of the T4 bacteriophage major head protein as the head protein emerges from the ribosome. This binding was discovered because EF-Tu bound to Gol peptide is the specific substrate of the Lit protease that cleaves the EF-Tu between amino acid residues Gly59 and lle60, blocking phage development. These experiments raised the question of why the Gol region of the incipient head protein binds to EF-Tu, as binding to incipient proteins is not expected from the canonical role of EF-Tu. Here, we use gol-lacZ translational fusions to show that cleavage of EF-Tu in the complex with Gol peptide can block translation of a lacZ reporter gene fused translationally downstream of the Gol peptide that activated the cleavage. We propose a model to explain how binding of EF-Tu to the emerging Gol peptide could cause translation to pause temporarily and allow time for the leader polypeptide to bind to the GroEL chaperonin before translation continues, allowing cotranslation of the head protein with its insertion into the GroEL chaperonin chamber, and preventing premature synthesis and precipitation of the head protein. Cleavage of EF-Tu in the complex would block translation of the head protein and therefore development of the infecting phage. Experiments are presented that confirm two predictions of this model. Considering the evolutionary conservation of the components of this system, this novel regulatory mechanism could be used in other situations, both in bacteria and eukaryotes, where proteins are cotranslated with their insertion into cellular structures.

• Benach J et al.
• The 2.3-A crystal structure of the shikimate 5-dehydrogenase orthologue YdiB from Escherichia coli suggests a novel catalytic environment for an NAD-dependent dehydrogenase.
• J Biol Chem. 2003; 278: 19176-82
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• We present here the 2.3-A crystal structure of the Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase. This enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been promoted as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the protomer contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue in close apposition to the nicotinamide ring and a clamp over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation in all orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction.

• Roth NJ, Penner RM, Huber RE
• Beta-galactosidases (Escherichia coli) with double substitutions show that Tyr-503 acts independently of Glu-461 but cooperatively with Glu-537.
• J Protein Chem. 2003; 22: 663-8
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• Beta-galactosidases with single substitutions for Tyr-503, Glu-461, and Glu-537 and with double substitutions for Tyr-503 and either Glu-461 or Glu-537 were constructed. Control experiments showed that the very low kcat values obtained for the double-substituted enzymes were not a result of contamination, reversion, or nonactive site activity catalyzed on the surface of the proteins. Circular dichroism studies showed that the structures of the enzymes were intact. E461Q/Y503F-beta-galactosidase was inactivated in an "additive" manner. This indicated that Glu-461 and Tyr-503 act independently in catalysis. Because these residues are at opposite sides of the active site and act in different steps, this is expected. E537D/Y503F-beta-galactosidase was only inactivated a few-fold more than the most inactive of its two single-substituted constituent beta-galactosidases. This showed that Glu-537 and Tyr-503 interact cooperatively on the same step. This correlates well with the proposed role of Tyr-503 as an acid catalyst for the breakage of the covalent bond between Glu-537 and galactose.

• Parkinson GN, Lee MP, Neidle S
• Crystal structure of parallel quadruplexes from human telomeric DNA.
• Nature. 2002; 417: 876-80
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• Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell from recombination and degradation. Disruption of telomere maintenance leads to eventual cell death, which can be exploited for therapeutic intervention in cancer. Telomeric DNA sequences can form four-stranded (quadruplex) structures, which may be involved in the structure of telomere ends. Here we describe the crystal structure of a quadruplex formed from four consecutive human telomeric DNA repeats and grown at a K(+) concentration that approximates its intracellular concentration. K(+) ions are observed in the structure. The folding and appearance of the DNA in this intramolecular quadruplex is fundamentally different from the published Na(+)-containing quadruplex structures. All four DNA strands are parallel, with the three linking trinucleotide loops positioned on the exterior of the quadruplex core, in a propeller-like arrangement. The adenine in each TTA linking trinucleotide loop is swung back so that it intercalates between the two thymines. This DNA structure suggests a straightforward path for telomere folding and unfolding, as well as ways in which it can recognize telomere-associated proteins.

• Canestro C, Albalat R, Escriva H, Gonzalez-Duarte R
• Endogenous beta-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system.
• Dev Genes Evol. 2001; 211: 154-6
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• Endogenous beta-galactosidase activity has been shown in the digestive tract of amphioxus from the larval to the adult stage and it can be easily followed as a histochemical marker. Enzymatic activity first appeared in 30-h larvae, became evident in 36-h larvae and remained in adults. In situ detection of beta-galactosidase activity was used to monitor morphological and functional differentiation of the digestive system and the posteriorization of the endodermal structures in retinoic-acid treated embryos. The endogenous beta-galactosidase activity was distinguished from the bacterial lacZ reporter by incubation at low pH.

• Matsumura I, Ellington AD
• In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates.
• J Mol Biol. 2001; 305: 331-9
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• The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity.

• Tanahashi H, Tabira T
• Alkaline treatment after X-Gal staining reaction for Escherichia coli beta-galactosidase enhances sensitivity.
• Anal Biochem. 2000; 279: 122-3

• Huber RE, Gaunt MT
• The inhibition of beta-galactosidase (Escherichia coli) by amino sugars and amino alcohols.
• Can J Biochem. 1982; 60: 608-12
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• Kinetically determined competitive inhibitor constants, which were estimates of the binding capacity of inhibitors interacting with the free enzyme form of beta-galactosidase (Escherichia coli), showed that amino sugars and alcohols inhibited the enzyme much more than did their respective parent sugars and alcohols. In most cases the inhibition was 10-30 times greater, but the inhibition by 1-aminogalactopyranose was about 300 times greater than that of galactopyranose. When the amino groups were acetylated the inhibitory advantage was completely eliminated and partial neutralization of the amino group of an inhibitor by the presence of a group with a negative charge on the same inhibitor decrease the inhibitory advantage. Studies of binding to the galactosyl form of the enzyme (rather than to the free form) indicated that the binding at the "glucose" site was increased only slightly by the presence of the amino group. Overall, the findings suggested that beta-galactosidase has a negative charge near the anomeric carbon binding position of the "galactose" site. Since negative charges are unlikely to be of any importance in binding the normally neutral beta-galactosidase substrates, this charge may be important in stabilizing a positively charged reaction analogous to an oxonium-carbonium ion intermediate.

• Welply JK, Fowler AV, Beckwith JR, Zabin I
• Positions of early nonsense and deletion mutations in lacZ.
• J Bacteriol. 1980; 142: 732-4
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• The positions of three Escherichia coli lacZ operator-proximal nonsense mutations and one deletion mutation have been determined. The nonsense mutations were suppressed with supF, resulting in the production of active beta-galactosidase by each strain. Amino acid sequencing identified the positions of the tyrosine residues inserted by supF, and thereby established that nonsense mutations lacZ2, lacZ2246, and lacZU131 are at sites corresponding to amino acids 23, 36, and 41 of beta-galactosidase, respectively. The deletion mutant, lacZM112, produced a dimeric beta-galactosidase protein missing amino acid residues 23 through 31 of the native enzyme.

• Sinnott ML
• Ions, ion-pairs and catalysis by the lacZ beta-galactosidase of Escherichia coli.
• FEBS Lett. 1978; 94: 1-9