SMART MODE:

NORMAL GENOMIC

• Garcia Garcia MI, Lau K, von Itzstein M, Garcia Carmona F, Sanchez Ferrer A
• Molecular characterization of a new N-acetylneuraminate synthase (NeuB1) from Idiomarina loihiensis.
• Glycobiology. 2015; 25: 115-23
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• N-Acetylneuraminate lyase synthase (NeuB; E.C. 2.5.1.56) is a key enzyme in pathogenic microorganisms for producing N-acetylneuraminic acid through the irreversible condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N-acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 (IlNeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40 degrees C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-beta-synthase domain (cystathionine-beta-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I-X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (>700 residues) NeuB, such as that existing in domain architectures 3 and 4.

• Klermund L, Groher A, Castiglione K
• New N-acyl-D-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate.
• J Biotechnol. 2013; 168: 256-63
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• N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-D-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-D-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-D-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117+/-2 U mg(-1) at 37 degrees C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the Km for the substrate than on the enzyme activity. At high substrate concentrations (>/=200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.

• Kalivoda KA, Steenbergen SM, Vimr ER
• Control of the Escherichia coli sialoregulon by transcriptional repressor NanR.
• J Bacteriol. 2013; 195: 4689-701
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• NanR, one of >8,500 GntR superfamily helix-turn-helix transcriptional regulators, controls expression of the genes required for catabolism of sialic acids in Escherichia coli. It is predicted to do the same in related bacteria harboring orthologs of nanR. The sialic acids are a family of over 40 naturally occurring nine-carbon keto-sugar acids found mainly in the animal lineage, which includes starfish to humans in the deuterostome lineage. Sialic acids function in development, immunity, protein localization and stability, and homeostasis. They also serve as microbial carbon and nitrogen sources and ligands for cell recognition during host colonization. The importance of microbial sialic acid metabolism for host-microbe interactions has made it a target for therapeutic development. Exploiting this target depends on understanding sialometabolic pathways in a wide range of evolutionarily distinct bacteria. Here, we show by transcriptome, genetic, and biochemical analyses that the most common sialic acid, N-acetylneuraminate, induces the nanATEK-yhcH, yjhATS (nanCMS), and yjhBC operons by directly inactivating NanR, converting the predominantly dimeric form of the repressor to an inactive monomer of approximately 30-kDa. Additionally, other results identify critical amino acid residues and nucleotides in the regulator and operator, respectively. The combined results better define how sialic acids, acting through NanR, affect the metabolic flux of an important group of host-derived metabolites. Thus, E. coli serves as a valuable model for understanding sialocatabolic pathways in bacteria.

• Pietrancosta N et al.
• Successful prediction of substrate-binding pocket in SLC17 transporter sialin.
• J Biol Chem. 2012; 287: 11489-97
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• Secondary active transporters from the SLC17 protein family are required for excitatory and purinergic synaptic transmission, sialic acid metabolism, and renal function, and several members are associated with inherited neurological or metabolic diseases. However, molecular tools to investigate their function or correct their genetic defects are limited or absent. Using structure-activity, homology modeling, molecular docking, and mutagenesis studies, we have located the substrate-binding site of sialin (SLC17A5), a lysosomal sialic acid exporter also recently implicated in exocytotic release of aspartate. Human sialin is defective in two inherited sialic acid storage diseases and is responsible for metabolic incorporation of the dietary nonhuman sialic acid N-glycolylneuraminic acid. We built cytosol-open and lumen-open three-dimensional models of sialin based on weak, but significant, sequence similarity with the glycerol-3-phosphate and fucose permeases from Escherichia coli, respectively. Molecular docking of 31 synthetic sialic acid analogues to both models was consistent with inhibition studies. Narrowing the sialic acid-binding site in the cytosol-open state by two phenylalanine to tyrosine mutations abrogated recognition of the most active analogue without impairing neuraminic acid transport. Moreover, a pilot virtual high-throughput screening of the cytosol-open model could identify a pseudopeptide competitive inhibitor showing >100-fold higher affinity than the natural substrate. This validated model of human sialin and sialin-guided models of other SLC17 transporters should pave the way for the identification of inhibitors, glycoengineering tools, pharmacological chaperones, and fluorescent false neurotransmitters targeted to these proteins.

• Bhaskar V, Kumar M, Manicka S, Tripathi S, Venkatraman A, Krishnaswamy S
• Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.
• Proteins. 2011; 79: 1132-42
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• YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.

• Chou CY et al.
• Modulation of substrate specificities of D-sialic acid aldolase through single mutations of Val-251.
• J Biol Chem. 2011; 286: 14057-64
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• In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RS-aldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (alpha/beta)(8)-barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase.l-arabinose and RS-aldolase.hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C(6) to C(10) for the design of antagonists and inhibitors of glycoenzymes.

• Sanchez-Carron G et al.
• Molecular characterization of a novel N-acetylneuraminate lyase from Lactobacillus plantarum WCFS1.
• Appl Environ Microbiol. 2011; 77: 2471-8
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• N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-d-mannosamine (ManNAc). In nature, N-acetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how an N-acetylneuraminate lyase was cloned from the human gut commensal Lactobacillus plantarum WCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter(-1) culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70 degrees C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed.

• Riedel TJ, Johnson LC, Knight J, Hantgan RR, Holmes RP, Lowther WT
• Structural and biochemical studies of human 4-hydroxy-2-oxoglutarate aldolase: implications for hydroxyproline metabolism in primary hyperoxaluria.
• PLoS One. 2011; 6: 26021-26021
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• BACKGROUND: 4-hydroxy-2-oxoglutarate (HOG) aldolase is a unique enzyme in the hydroxyproline degradation pathway catalyzing the cleavage of HOG to pyruvate and glyoxylate. Mutations in this enzyme are believed to be associated with the excessive production of oxalate in primary hyperoxaluria type 3 (PH3), although no experimental data is available to support this hypothesis. Moreover, the identity, oligomeric state, enzymatic activity, and crystal structure of human HOGA have not been experimentally determined. METHODOLOGY/PRINCIPAL FINDINGS: In this study human HOGA (hHOGA) was identified by mass spectrometry of the mitochondrial enzyme purified from bovine kidney. hHOGA performs a retro-aldol cleavage reaction reminiscent of the trimeric 2-keto-3-deoxy-6-phosphogluconate aldolases. Sequence comparisons, however, show that HOGA is related to the tetrameric, bacterial dihydrodipicolinate synthases, but the reaction direction is reversed. The 1.97 A resolution crystal structure of hHOGA bound to pyruvate was determined and enabled the modeling of the HOG-Schiff base intermediate and the identification of active site residues. Kinetic analyses of site-directed mutants support the importance of Lys196 as the nucleophile, Tyr168 and Ser77 as components of a proton relay, and Asn78 and Ser198 as unique residues that facilitate substrate binding. CONCLUSIONS/SIGNIFICANCE: The biochemical and structural data presented support that hHOGA utilizes a type I aldolase reaction mechanism, but employs novel residue interactions for substrate binding. A mapping of the PH3 mutations identifies potential rearrangements in either the active site or the tetrameric assembly that would likely cause a loss in activity. Altogether, these data establish a foundation to assess mutant forms of hHOGA and how their activity could be pharmacologically restored.

• Devenish SR, Gerrard JA
• The quaternary structure of Escherichia coli N-acetylneuraminate lyase is essential for functional expression.
• Biochem Biophys Res Commun. 2009; 388: 107-11
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• As part of a general study into the impact of quaternary structure on enzyme function, a library of 31 point mutations were engineered at the dimer-dimer interface of the homotetrameric (beta/alpha)(8)-barrel protein, N-acetylneuraminate lyase (NAL, EC 4.1.3.3). Disruption of the interface generated either soluble tetramers or putative dimers that were absolutely insoluble and inactive. Intriguingly, the soluble tetramers were found to have widely varying k(cat) values, hinting at a role for the interface in catalysis. Leucine 171 was identified as essential to interface integrity. We conclude that the dimer-dimer interface of NAL is intolerant to mutation and essential for functional expression.

• Steenbergen SM, Jirik JL, Vimr ER
• YjhS (NanS) is required for Escherichia coli to grow on 9-O-acetylated N-acetylneuraminic acid.
• J Bacteriol. 2009; 191: 7134-9
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• The nanATEK-yhcH, yjhATS, and yjhBC operons in Escherichia coli are coregulated by environmental N-acetylneuraminic acid, the most prevalent sialic acid in nature. Here we show that YjhS (NanS) is a probable 9-O-acetyl N-acetylneuraminic acid esterase required for E. coli to grow on this alternative sialic acid, which is commonly found in mammalian host mucosal sites.

• He SM, Luo Y, Hove-Jensen B, Zechel DL
• A fluorescent substrate for carbon-phosphorus lyase: towards the pathway for organophosphonate metabolism in bacteria.
• Bioorg Med Chem Lett. 2009; 19: 5954-7
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• Many species of bacteria can use naturally occurring organophosphonates as a source of metabolic phosphate by cleaving the carbon-phosphorus bond with a multi-enzyme pathway collectively called carbon-phosphorus lyase (CP-lyase). Very little is known about the fate of organophosphonates entering this pathway. In order to detect metabolic intermediates we have synthesized a fluorescently labelled organophosphonate and show that this is a viable substrate for the CP-lyase pathway in Escherichia coli and that the expected product of CP-bond cleavage is formed. The in vivo competence of one potential metabolic intermediate, 1-ethylphosphonate-alpha-D-ribofuranose, is also demonstrated.

• Kefala G et al.
• Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis.
• Biochem J. 2008; 411: 351-60
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• The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 A (1 A=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (beta/alpha)(8)-barrel followed by a C-terminal alpha-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (beta/alpha)(8)-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr(143), Thr(54) and Tyr(117) appears to be disrupted. The kinetic parameters of the enzyme are reported: K(M)(ASA)=0.43+/-0.02 mM, K(M)(pyruvate)=0.17+/-0.01 mM and V(max)=4.42+/-0.08 micromol x s(-1) x mg(-1). Interestingly, the V(max) of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

• Fullerton SW et al.
• Mechanism of the Class I KDPG aldolase.
• Bioorg Med Chem. 2006; 14: 3002-10
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• In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.

• Zaitseva J, Lu J, Olechoski KL, Lamb AL
• Two crystal structures of the isochorismate pyruvate lyase from Pseudomonas aeruginosa.
• J Biol Chem. 2006; 281: 33441-9
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• Enzymatic systems that exploit pericyclic reaction mechanisms are rare. A recent addition to this class is the enzyme PchB, an 11.4-kDa isochorismate pyruvate lyase from Pseudomonas aeruginosa. The apo and pyruvate-bound structures of PchB reveal that the enzyme is a structural homologue of chorismate mutases in the AroQalpha class despite low sequence identity (20%). The enzyme is an intertwined dimer of three helices with connecting loops, and amino acids from each monomer participate in each of two active sites. The apo structure (2.35 A resolution) has one dimer per asymmetric unit with nitrate bound in an open active site. The loop between the first and second helices is disordered, providing a gateway for substrate entry and product exit. The pyruvate-bound structure (1.95 A resolution) has two dimers per asymmetric unit. One has two open active sites like the apo structure, and the other has two closed active sites with the loop between the first and second helices ordered for catalysis. Determining the structure of PchB is part of a larger effort to elucidate protein structures involved in siderophore biosynthesis, as these enzymes are crucial for bacterial iron uptake and virulence and have been identified as antimicrobial drug targets.

• Gorelik M, Lunin VV, Skarina T, Savchenko A
• Structural characterization of GntR/HutC family signaling domain.
• Protein Sci. 2006; 15: 1506-11
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• The crystal structure of Escherichia coli PhnF C-terminal domain (C-PhnF) was solved at 1.7 A resolution by the single wavelength anomalous dispersion (SAD) method. The PhnF protein belongs to the HutC subfamily of the large GntR transcriptional regulator family. Members of this family share similar N-terminal DNA-binding domains, but are divided into four subfamilies according to their heterogenic C-terminal domains, which are involved in effector binding and oligomerization. The C-PhnF structure provides for the first time the scaffold of this domain for the HutC subfamily, which covers about 31% of GntR-like regulators. The structure represents a mixture of alpha-helices and beta-strands, with a six-stranded antiparallel beta-sheet at the core. C-PhnF monomers form a dimer by establishing interdomain eight-strand beta-sheets that include core antiparallel and N-terminal two-strand parallel beta-sheets from each monomer. C-PhnF shares strong structural similarity with the chorismate lyase fold, which features a buried active site locked behind two helix-turn-helix loops. The structural comparison of the C-PhnF and UbiC proteins allows us to propose that a similar site in the PhnF structure is adapted for effector binding.

• Williams GJ, Woodhall T, Nelson A, Berry A
• Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics.
• Protein Eng Des Sel. 2005; 18: 239-46
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• Analogues of N-acetylneuraminic acid (sialic acid, NANA, Neu5Ac), including 6-dipropylcarboxamides, have been found to be selective and potent inhibitors of influenza sialidases. Sialic acid analogues are, however, difficult to synthesize by traditional chemical methods and the enzyme N-acetylneuraminic acid lyase (NAL) has previously been used for the synthesis of a number of analogues. The activity of this enzyme towards 6-dipropylcarboxamides is, however, low. Here, we used structure-guided saturation mutagenesis to produce variants of NAL with improved activity and specificity towards 6-dipropylcarboxamides. Three residues were targeted for mutagenesis, Asp191, Glu192 and Ser208. Only substitution at position 192 produced significant improvements in activity towards the dipropylamide. One variant, E192N, showed a 49-fold improvement in catalytic efficiency towards the target analogue and a 690-fold shift in specificity from sialic acid towards the analogue. These engineering efforts provide a scaffold for the further tailoring of NAL for the synthesis of sialic acid mimetics.

• Schofield LR, Anderson BF, Patchett ML, Norris GE, Jameson GB, Parker EJ
• Substrate ambiguity and crystal structure of Pyrococcus furiosus 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase: an ancestral 3-deoxyald-2-ulosonate-phosphate synthase?
• Biochemistry. 2005; 44: 11950-62
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• 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAH7PS) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and the four-carbon monosaccharide D-erythrose 4-phosphate (E4P). DAH7PS from the hyperthermophile Pyrococcus furiosus is a member of the DAH7PS Ibeta subfamily, which also includes the KDO8PS enzymes. KDO8PS (3-deoxy-D-manno-octulosonate-8-phosphate synthase) catalyzes a closely related reaction of PEP with the five-carbon monosaccharide D-arabinose 5-phosphate (A5P). DAH7PS from P. furiosus requires a metal ion for activity and, unlike other characterized DAH7PS enzymes, is not inhibited by aromatic amino acids. Purified P. furiosus DAH7PS is able to utilize not only the four-carbon phosphorylated monosaccharides E4P and 2-deoxy-D-erythrose 4-phosphate but also the five-carbon phosphorylated monosaccharides A5P, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate with similar kcat but much increased KM values. DL-glyceraldehyde 3-phosphate and D-glucose 6-phosphate are not substrates. The structure of recombinant P. furiosus DAH7PS in complex with PEP was determined to 2.25 A resolution. The asymmetric unit consists of a dimer of (beta/alpha)8-barrel subunits. Analysis of the buried surfaces formed by dimerization and tetramerization, as observed in the crystal structure, provides insight into both the oligomeric status in solution and the substrate ambiguity of P. furiosus DAH7PS. P. furiosus DAH7PS is both the first archaeal and the first "naked" DAH7PS (without N-terminal extensions) to be fully characterized functionally and structurally. The broad substrate specificity of this DAH7PS, the lack of allosteric inhibition, and various structural features indicate that, of the enzymes characterized to date, P. furiosus DAH7PS may be the contemporary protein closest to the ancestral type I enzyme.

• 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.

• Teplyakov A, Obmolova G, Toedt J, Galperin MY, Gilliland GL
• Crystal structure of the bacterial YhcH protein indicates a role in sialic acid catabolism.
• J Bacteriol. 2005; 187: 5520-7
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• The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-A resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded beta-helix. Two antiparallel beta-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.

• Lokanath NK et al.
• Structure of aldolase from Thermus thermophilus HB8 showing the contribution of oligomeric state to thermostability.
• Acta Crystallogr D Biol Crystallogr. 2004; 60: 1816-23
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• 2-Deoxyribose-5-phosphate aldolase catalyzes a reversible aldol condensation of two aldehydes via formation of a covalent Schiff-base intermediate at the active lysine residue. The crystal structure of 2-deoxyribose-5-phosphate aldolase from Thermus thermophilus HB8 has been determined with and without the substrate at atomic resolution. This enzyme, which has a unique homotetramer structure, has been compared with the previously reported crystal structures of two orthologues from Escherichia coli and Aeropyrum pernix. In contrast to the similar alpha/beta-barrel fold of the monomers, substantial quaternary structural differences are observed between these three enzymes. Further comparison of the subunit-subunit interface areas of these aldolases showed a clear positive correlation between the interface area and the living temperature of the source organism. From these results, it is concluded that the oligomeric state of 2-deoxyribose-5-phosphate aldolase is important for the thermostability and not for the catalytic function.

• Suryanti V, Nelson A, Berry A
• Cloning, over-expression, purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae.
• Protein Expr Purif. 2003; 27: 346-56
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• N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues.

• Kroemer M, Merkel I, Schulz GE
• Structure and catalytic mechanism of L-rhamnulose-1-phosphate aldolase.
• Biochemistry. 2003; 42: 10560-8
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• The structure of L-rhamnulose-1-phosphate aldolase has been established at 1.35 A resolution in a crystal form that was obtained by a surface mutation and has one subunit of the C(4)-symmetric tetramer in the asymmetric unit. It confirms an earlier 2.7 A resolution structure which was determined in a complicated crystal form with 20 subunits per asymmetric unit. The chain fold and the active center are similar to those of L-fuculose-1-phosphate aldolase and L-ribulose-5-phosphate 4-epimerase. The active center similarity is supported by a structural comparison of all three enzymes and by the binding mode of the inhibitor phosphoglycolohydroxamate at the site of the product dihydroxyacetone phosphate for the two aldolases. The sensitivity of the catalytic rate to several mutations and a comparison with the established mechanism of the related aldolase give rise to a putative catalytic mechanism. This mechanism involves the same binding mode of the second product L-lactaldehyde in both aldolases, except for a 180 degrees flip of the aldehyde group distinguishing between the two epimers rhamnulose and fuculose. The N-terminal domain exhibits a correlated anisotropic mobility that channels the isotropic Brownian motion into a directed movement of the catalytic base and the substrate phosphate on the N-domain toward the zinc ion and the lactaldehyde on the C-terminal domain. We suggest that this movement supports the catalysis mechanically.

• Marco-Marin C, Ramon-Maiques S, Tavarez S, Rubio V
• Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase.
• J Mol Biol. 2003; 334: 459-76
• Display abstract

• We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings.

• Joerger AC, Mayer S, Fersht AR
• Mimicking natural evolution in vitro: an N-acetylneuraminate lyase mutant with an increased dihydrodipicolinate synthase activity.
• Proc Natl Acad Sci U S A. 2003; 100: 5694-9
• Display abstract

• N-acetylneuraminate lyase (NAL) and dihydrodipicolinate synthase (DHDPS) belong to the NAL subfamily of (betaalpha)(8)-barrels. They share a common catalytic step but catalyze reactions in different biological pathways. By rational design, we have introduced various mutations into the NAL scaffold from Escherichia coli to switch the activity toward DHDPS. These mutants were tested with respect to their catalytic properties in vivo and in vitro as well as their stability. One point mutation (L142R) was sufficient to create an enzyme that could complement a bacterial auxotroph lacking the gene for DHDPS as efficiently as DHDPS itself. In vitro, this mutant had an increased DHDPS activity of up to 19-fold as defined by the specificity constant k(cat)K(M) for the new substrate l-aspartate-beta-semialdehyde when compared with the residual activity of NAL wild-type, mainly because of an increased turnover rate. At the same time, mutant L142R maintained much of its original NAL activity. We have solved the crystal structure of mutant L142R at 1.8 A resolution in complex with the inhibitor beta-hydroxypyruvate. This structure reveals that the conformations of neighboring active site residues are left virtually unchanged by the mutation. The high flexibility of R142 may favor its role in assisting in catalysis. Perhaps, nature has exploited the catalytic promiscuity of many enzymes to evolve novel enzymes or biological pathways during the course of evolution.

• Hwang TS et al.
• Structural characterization of Escherichia coli sialic acid synthase.
• Biochem Biophys Res Commun. 2002; 295: 167-73
• Display abstract

• Sialic acid synthase encoded by the neuB gene of Escherichia coli catalyzes the condensation of N-acetylmannosamine and phosphoenolpyruvate to form N-acetylneuraminic acid. This report demonstrates the first structural information on sialic acid synthase by CD, MALDI-TOF, and chemical cross-linking studies. Also, a specific cleavage by endogenous protease(s) has been identified at Lys(280) of the enzyme (40 kDa) by LC-MS and N-terminal sequencing analyses. The cleavage results in the formation of two inactive fragments of 33 and 7 kDa. The structural analysis indicates that the fragmentation is associated with a significant change of the enzyme from a tetrameric to trimeric form, and alterations in both secondary and native quaternary structures.

• Thorell S, Schurmann M, Sprenger GA, Schneider G
• Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family.
• J Mol Biol. 2002; 319: 161-71
• Display abstract

• Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.

• Gallagher DT, Mayhew M, Holden MJ, Howard A, Kim KJ, Vilker VL
• The crystal structure of chorismate lyase shows a new fold and a tightly retained product.
• Proteins. 2001; 44: 304-11
• Display abstract

• The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type. The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate. Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.

• Luo Y, Samuel J, Mosimann SC, Lee JE, Tanner ME, Strynadka NC
• The structure of L-ribulose-5-phosphate 4-epimerase: an aldolase-like platform for epimerization.
• Biochemistry. 2001; 40: 14763-71
• Display abstract

• The structure of L-ribulose-5-phosphate 4-epimerase from E. coli has been solved to 2.4 A resolution using X-ray diffraction data. The structure is homo-tetrameric and displays C(4) symmetry. Each subunit has a single domain comprised of a central beta-sheet flanked on either side by layers of alpha-helices. The active site is identified by the position of the catalytic zinc residue and is located at the interface between two adjacent subunits. A remarkable feature of the structure is that it shows a very close resemblance to that of L-fuculose-1-phosphate aldolase. This is consistent with the notion that both enzymes belong to a superfamily of epimerases/aldolases that catalyze carbon-carbon bond cleavage reactions via a metal-stabilized enolate intermediate. Detailed inspection of the epimerase structure, however, indicates that despite the close overall structural similarity to class II aldolases, the enzyme has evolved distinct active site features that promote its particular chemistry.

• Kruger D, Schauer R, Traving C
• Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens: insights into the reaction mechanism.
• Eur J Biochem. 2001; 268: 3831-9
• Display abstract

• The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography. The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 degrees C. In kinetic studies the lyase exhibits a Km of 3.2 mM for Neu5Ac and a Vmax of 27.5 U x mg(-1). To clarify the functional role of some putative active site residues, site-directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate. Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding. In this respect the carboxy group of aspartate 187 seems to be particularly important. Based on the results of these studies, a model of the reaction mechanism is discussed.

• 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
• Display abstract

• 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.

• Abergel C et al.
• Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 A resolution.
• Structure. 1999; 7: 1291-300
• Display abstract

• BACKGROUND: The periplasmic protein TolB from Escherichia coli is part of the Tol-PAL (peptidoglycan-associated lipoprotein) multiprotein complex used by group A colicins to penetrate and kill cells. TolB homologues are found in many gram-negative bacteria and the Tol-PAL system is thought to play a role in bacterial envelope integrity. TolB is required for lethal infection by Salmonella typhimurium in mice. RESULTS: The crystal structure of the selenomethionine-substituted TolB protein from E. coli was solved using multiwavelength anomalous dispersion methods and refined to 1. 95 A. TolB has a two-domain structure. The N-terminal domain consists of two alpha helices, a five-stranded beta-sheet floor and a long loop at the back of this floor. The C-terminal domain is a six-bladed beta propeller. The small, possibly mobile, contact area (430 A(2)) between the two domains involves residues from the two helices and the first and sixth blades of the beta propeller. All available genomic sequences were used to identify new TolB homologues in gram-negative bacteria. The TolB structure was then interpreted using the observed conservation pattern. CONCLUSIONS: The TolB beta-propeller C-terminal domain exhibits sequence similarities to numerous members of the prolyl oligopeptidase family and, to a lesser extent, to class B metallo-beta-lactamases. The alpha/beta N-terminal domain shares a structural similarity with the C-terminal domain of transfer RNA ligases. We suggest that the TolB protein might be part of a multiprotein complex involved in the recycling of peptidoglycan or in its covalent linking with lipoproteins.

• Revilla-Nuin B, Rodriguez-Aparicio LB, Ferrero MA, Reglero A
• Regulation of capsular polysialic acid biosynthesis by N-acetyl-D-mannosamine, an intermediate of sialic acid metabolism.
• FEBS Lett. 1998; 426: 191-5
• Display abstract

• N-Acetyl-D-mannosamine (ManNAc) is a specific substrate for the synthesis of N-acetylneuraminic acid, the essential precursor of bacterial capsular polysialic acid (PA). When Escherichia coli K92 used ManNAc as a carbon source, we observed a dramatic reduction (up to 90%) in in vivo PA production. Experiments in which the carbon source was changed revealed that the maximal inhibitory effect occurred when this sugar was present in the medium before the logarithmic phase of bacterial growth had started. Enzymatic analysis revealed that high concentrations of ManNAc-6-phosphate inhibit NeuAc lyase, the enzyme that synthesizes NeuAc for PA biosynthesis in E. coli. These results indicate that ManNAc-6-phosphate is able to regulate NeuAc lyase activity and modulate the PA synthesis.

• Jia J et al.
• Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the alpha/beta barrel within the class I aldolase family.
• Structure. 1996; 4: 715-24
• Display abstract

• BACKGROUND: Transaldolase is one of the enzymes in the non-oxidative branch of the pentose phosphate pathway. It transfers a C3 ketol fragment from a ketose donor to an aldose acceptor. Transaldolase, together with transketolase, creates a reversible link between the pentose phosphate pathway and glycolysis. The enzyme is of considerable interest as a catalyst in stereospecific organic synthesis and the aim of this work was to reveal the molecular architecture of transaldolase and provide insights into the structural basis of the enzymatic mechanism. RESULTS: The three-dimensional (3D) structure of recombinant transaldolase B from E. coli was determined at 1.87 A resolution. The enzyme subunit consists of a single eight-stranded alpha/beta-barrel domain. Two subunits form a dimer related by a twofold symmetry axis. The active-site residue Lys132 which forms a Schiff base with the substrate is located at the bottom of the active-site cleft. CONCLUSIONS: The 3D structure of transaldolase is similar to structures of other enzymes in the class I aldolase family. Comparison of these structures suggests that a circular permutation of the protein sequence might have occurred in transaldolase, which nevertheless results in a similar 3D structure. This observation provides evidence for a naturally occurring circular permutation in an alpha/beta-barrel protein. It appears that such genetic permutations occur more frequently during evolution than was previously thought.

• Janecek S
• Similarity of different beta-strands flanked in loops by glycines and prolines from distinct (alpha/beta)8-barrel enzymes: chance or a homology?
• Protein Sci. 1995; 4: 1239-42
• Display abstract

• Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed.

• 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
• Display abstract

• 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.

• 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
• Display abstract

• 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.

• Uhlin U, Eklund H
• Structure of ribonucleotide reductase protein R1.
• Nature. 1994; 370: 533-9
• Display abstract

• Ribonucleotide reductase is the only enzyme that catalyses de novo formation of deoxyribonucleotides and is thus a key enzyme in DNA synthesis. The radical-based reaction involves five cysteins. Two redox-active cysteines are located at adjacent antiparallel strands in a new type of ten-stranded alpha/beta-barrel, and two others at the carboxyl end in a flexible arm. The fifth cysteine, in a loop in the centre of the barrel, is positioned to initiate the radical reaction.

• Knibbs RN, Osborne SE, Glick GD, Goldstein IJ
• Binding determinants of the sialic acid-specific lectin from the slug Limax flavus.
• J Biol Chem. 1993; 268: 18524-31
• Display abstract

• The specific structural features of 24 N-acetylneuraminic acid derivatives required for the high affinity interaction of sialoglycoconjugates with the sialic acid-specific lectin from the slug Limax flavus were studied by hapten inhibition of precipitation. These results provide insight regarding the structure of the binding pocket for N-acetylneuraminic acid that exists in L. flavus agglutinin (LFA). The alpha-anomer of sialic acid is a very important factor in binding to the slug lectin. The carboxylic acid group makes only a moderate contribution to binding, since modifications of the carboxylic group decrease binding approximately 5-fold. Modification or removal of the hydroxyl group on carbon 4 does not affect binding. However, when the C4 epimer was tested, there was a dramatic decrease in binding, suggesting that whereas the equatorial hydroxyl at C4 does not contribute to binding, the introduction of an axial hydroxyl group at C4 sterically hinders the binding interaction. The substituent on the 5-amino group occupies an important role in binding of Neu5Ac to LFA as well. When the acetyl is modified by the addition of a hydroxyl group to yield the N-glycolyl derivative, we observed a 20-fold decrease, while the removal of the methyl to form the N-formyl derivative resulted in a 50-fold decrease. The 5-amino derivative was the poorest inhibitor of all compounds examined, indicating a critical role for the N-acetyl group in high affinity binding to LFA. The glyceryl tail also appears to be critical for binding inasmuch as acetylation of the C9 hydroxyl group or periodate cleavage of carbons 8 and 9 resulted in a 20- to 50-fold decrease in binding. The equilibrium constant (K(a)) for binding of Neu5Ac to LFA is 3.8 x 10(4) M-1, with a single binding site (n = 0.85) per monomer.

• Vanni P, Giachetti E, Pinzauti G, McFadden BA
• Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme.
• Comp Biochem Physiol B. 1990; 95: 431-58

• Kondo A, Suzuki J, Kuraya N, Hase S, Kato I, Ikenaka T
• Improved method for fluorescence labeling of sugar chains with sialic acid residues.
• Agric Biol Chem. 1990; 54: 2169-70

• Kolisis FN, Evangelopoulos AE
• Studies on metabolic regulation and estimation of sialic acids by enzyme immobilization techniques.
• Prog Clin Biol Res. 1988; 259: 413-20

• Kawakami B, Kudo T, Narahashi Y, Horikoshi K
• Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene.
• J Bacteriol. 1986; 167: 404-6
• Display abstract

• Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.

• Kolisis FN
• Number and function of sulphydryl groups of N-acetylneuraminate lyase.
• Arch Int Physiol Biochim. 1985; 93: 89-92
• Display abstract

• The reaction between DTNB and the SH groups of N-acetylneuraminate lyase has been investigated in the presence and absence of pyruvic acid, substrate of the enzyme. It was found that DTNB inactivates N-acetylneuraminate lyase, while pyruvic acid protects the enzyme against this inactivation. When the enzyme was fully inactivated, two SH groups have reacted with DTNB. This result supports previous suggestions, that there is one cystein residue per active site responsible for enzyme activity. In the presence of SDS, approx. 6 SH groups reacted with DTNB suggesting the existence of 3 SH groups per enzyme subunit.

• Kakehi K, Maeda K, Teramae M, Honda S, Takai T
• Analysis of sialic acids by gas chromatography of the mannosamine derivatives released by the action of N-acetylneuraminate lyase.
• J Chromatogr. 1983; 272: 1-8
• Display abstract

• A convenient method for the analysis of sialic acids is proposed, which is based on their dissociation into pyruvate and N-acylmannosamines by the action of N-acetylneuraminate lyase, followed by gas-chromatographic analysis of the latter products as trimethylsilylated diethyl dithioacetals. Conjugated sialic acids should be freed with neuraminidase before being subjected to the action of the lyase, but these sequential enzymic reactions may be performed in one pot. N-Acetyl-, N-glycolyl- and N,O-diacetylneuraminic acids gave the corresponding mannosamines, and the dithioacetal derivatives of these mannosamines were well separated on a column of silicone OV-1. Quantitation of this enzymic and gas chromatographic method indicated that the error and coefficient of variation for free N-acetylneuraminic acid were 1.1% and 2.5%, respectively, for ten determinations at the 100 nmol level. The values for conjugated N-acetylneuraminic acid in N-acetylneuraminlactose were 2.9% and 5.9%, respectively. This method was applied to the analysis of sialic acids in some biological samples, and the results were compared with those obtained by the conventional colorimetric method. Preliminary data on urinary sialic acids indicated that cancerous patients gave significantly higher levels of urinary N-acetylneuraminic acid than normal subjects.

• Wang JK, Dekker EE, Lewinski ND, Winter HC
• Physical and chemical evidence for the trimeric subunit structure of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli K-12.
• J Biol Chem. 1981; 256: 1793-800

• Cabezas JA, Porto E
• [Sialic acids. 13. N-acetylneuraminic acid, hexosamine, and pyruvic acid contents and N-acetyl beta-glucosaminidase activity of normal and cancerous sera].
• Rev Esp Fisiol. 1970; 26: 339-45

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