Utilization of L-ascorbate by Escherichia coli K-12: assignments of functions to products of the yjf-sga and yia-sgb operons.

Escherichia coli K-12 can ferment L-ascorbate. The operon encoding catabolic enzymes in the utilization of L-ascorbate (ula) has been identified; this operon of previously unknown function had been designated the yif-sga operon. Three enzymes in the pathway that produce D-xylulose 5-phosphate have been functionally characterized: 3-keto-L-gulonate 6-phosphate decarboxylase (UlaD), L-xylulose 5-phosphate 3-epimerase (UlaE), and L-ribulose 5-phosphate 4-epimerase (UlaF). Several products of the yia-sgb operon were also functionally characterized, although the substrate and physiological function of the operon remain unknown: 2,3-diketo-L-gulonate reductase (YiaK), 3-keto-L-gulonate kinase (LyxK), 3-keto-L-gulonate 6-phosphate decarboxylase (SgbH), and L-ribulose 5-phosphate 4-epimerase (SgbE).     

Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts

Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization.     

The central cavity from the (alpha/alpha)6 barrel structure of Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase contains two key histidine residues for reversible conversion

N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) catalyzes the reversible epimerization between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc). We report here the 2.0 A resolution crystal structure of the GlcNAc 2-epimerase from Anabaena sp. CH1. The structure demonstrates an (alpha/alpha)(6) barrel fold, which shows structural homology with porcine GlcNAc 2-epimerase as well as a number of glycoside hydrolase enzymes and other sugar-metabolizing enzymes. One side of the barrel structure consists of short loops involved in dimer interactions. The other side of the barrel structure is comprised of long loops containing six short beta-sheets, which enclose a putative central active-site pocket. Site-directed mutagenesis of conserved residues near the N-terminal region of the inner alpha helices shows that R57, H239, E308, and H372 are strictly required for activity. E242 and R375 are also essential in catalysis. Based on the structure and kinetic analysis, H239 and H372 may serve as the key active site acid/base catalysts. These results suggest that the (alpha/alpha)(6) barrel represents a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner alpha helices, thus forming a fine-tuned catalytic site in GlcNAc 2-epimerase.     

Cloning and sequencing of the cellobiose 2-epimerase gene from an obligatory anaerobe, Ruminococcus albus

Cellobiose 2-epimerase (EC 5.1.3.11) was first identified in 1967 as an extracellular enzyme that catalyzes the reversible epimerization between cellobiose and 4-O-beta-D-glucopyranosyl-D-mannose in a culture broth of Ruminococcus albus 7 (ATCC 27210(T)). Here, for the first time, we describe the purification of cellobiose 2-epimerase from R. albus NE1. The enzyme was found to 2-epimerize the reducing terminal glucose moieties of cellotriose and cellotetraose in addition to cellobiose. The gene encoding cellobiose 2-epimerase comprises 1170 bp (389 amino acids) and is present as a single copy in the genome. The deduced amino acid sequence of the mature enzyme contains the possible catalytic residues Arg52, His243, Glu246, and His374. Sequence analysis shows the gene shares a very low level of homology with N-acetyl-D-glucosamine 2-epimerases (EC 5.1.3.8), but no significant homology to any other epimerases reported to date.     

Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol

In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an 18O-labeling study that demonstrates the synthase operates via a C-O bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (beta/alpha)8 barrel (TIM barrel)-type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 A resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the N-acetylmannosamine carbonyl to the addition of PEP.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

Dipole moment in TIM alpha/beta fold proteins

TIM proteins of alpha/beta barrel fold from alpha/beta class as given in SCOP database were taken for dipole moment analysis. In all, 32 structures were analyzed for their dipole moment contributions. Representative structures from 20 super families in the alpha/beta fold, with different enzyme functions and 12 protein domains of TIM family in TIM super family were considered. The active sites of these proteins are located on the C-terminal side of the beta-strands. The molecules of same alpha/beta fold, but differing in their functionality also showed a common electrostatic field pattern along the barrel axis and had the dipole moment along the barrel axis and towards C-terminal end of the beta-strands. However, it is observed from our calculations that the dipole moment direction is possibly a consequence of the structural fold, with distribution of charges playing a modulatory role, and does not contribute to the location of active site. We show here that apart from the commonly held view as proposed by Hol et al [Hol W G L, van Duijnen PT and Berendsen H J C (1978) Nature (London), 273, 443-446] of the role of the alpha helical dipole moment, the beta-sheets in the barrel can also have a considerable dipole moment contribution. Taken together with our dipole moment analysis on integral membrane proteins [Vasanthi G and Krishnaswamy S (2002) Indian J Biochem Biophys 39, 93-100], this suggests the need to examine the role of dipole moment in the case of especially beta sheets forming barrels.     

The crystal structure of methylglyoxal synthase from Escherichia coli.

BACKGROUND: The reaction mechanism of methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase (TIM). Both enzymes utilise dihydroxyacetone phosphate (DHAP) to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways. However, the second catalytic step in the MGS reaction pathway is characterized by the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal instead of reprotonation to form the isomer glyceraldehyde 3-phosphate. RESULTS: The crystal structure of MGS bound to formate and substoichiometric amounts of phosphate in the space group P6522 has been determined at 1.9 A resolution. This structure shows that the enzyme is a homohexamer composed of interacting five-stranded beta/alpha proteins, rather than the hallmark alpha/beta barrel structure of TIM. The conserved residues His19, Asp71, and His98 in each of the three monomers in the asymmetric unit bind to a formate ion that is present in the crystallization conditions. Differences in the three monomers in the asymmetric unit are localized at the mouth of the active site and can be ascribed to the presence or absence of a bound phosphate ion. CONCLUSIONS: In agreement with site-directed mutagenesis and mechanistic enzymology, the structure suggests that Asp71 acts as the catalytic base. Further, Asp20 and Asp101 are involved in intersubunit salt bridges. These salt bridges may provide a pathway for transmitting allosteric information.     

Structural basis for substrate specificity in phosphate binding (beta/alpha)8-barrels: D-allulose 6-phosphate 3-epimerase from Escherichia coli K-12

Enzymes that share the (beta/alpha) 8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal (beta/alpha) 2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth beta-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, DeltaT196, DeltaS197 and DeltaG198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in k cat/ K m are dominated by changes in k cat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth beta-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.     

Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (beta/alpha)8 barrel joins two active sites

BACKGROUND: Imidazole glycerol phosphate synthase catalyzes a two-step reaction of histidine biosynthesis at the bifurcation point with the purine de novo pathway. The enzyme is a new example of intermediate channeling by glutamine amidotransferases in which ammonia generated by hydrolysis of glutamine is channeled to a second active site where it acts as a nucleophile. In this case, ammonia reacts in a cyclase domain to produce imidazole glycerol phosphate and an intermediate of purine biosynthesis. The enzyme is also a potential target for drug and herbicide development since the histidine pathway does not occur in mammals. RESULTS: The 2.1 A crystal structure of imidazole glycerol phosphate synthase from yeast reveals extensive interaction of the glutaminase and cyclase catalytic domains. At the domain interface, the glutaminase active site points into the bottom of the (beta/alpha)(8) barrel of the cyclase domain. An ammonia tunnel through the (beta/alpha)(8) barrel connects the glutaminase docking site at the bottom to the cyclase active site at the top. A conserved "gate" of four charged residues controls access to the tunnel. CONCLUSIONS: This is the first structure in which all the components of the ubiquitous (beta/alpha)(8) barrel fold, top, bottom, and interior, take part in enzymatic function. Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site. The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity.     

A catalytic mechanism revealed by the crystal structures of the imidazolonepropionase from Bacillus subtilis

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-l-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.     

Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes

D-psicose, a rare sugar produced by the enzymatic reaction of D-tagatose 3-epimerase (DTEase), has been used extensively for the bioproduction of various rare carbohydrates. Recently characterized D-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was found to belong to the DTEase family and to catalyze the interconversion of D-fructose and D-psicose by epimerizing the C-3 position, with marked efficiency for D-psicose. The crystal structures of DPEase and its complex with the true substrate D-fructose were determined; DPEase is a tetramer and each monomer belongs to a TIM-barrel fold. The active site in each subunit is distinct from that of other TIM-barrel enzymes, which use phosphorylated ligands as the substrate. It contains a metal ion with octahedral coordination to two water molecules and four residues that are absolutely conserved across the DTEase family. Upon binding of D-fructose, the substrate displaces water molecules in the active site, with a conformation mimicking the intermediate cis-enediolate. Subsequently, Trp112 and Pro113 in the beta4-alpha4 loop undergo significant structural changes, sealing off the active site. Structural evidence and site-directed mutagenesis of the putative catalytic residues suggest that the metal ion plays a pivotal role in catalysis by anchoring the bound D-fructose, and Glu150 and Glu244 carry out an epimerization reaction at the C-3 position.     

Analysis of the class I aldolase binding site architecture based on the crystal structure of 2-deoxyribose-5-phosphate aldolase at 0.99A resolution

The crystal structure of the bacterial (Escherichia coli) class I 2-deoxyribose-5-phosphate aldolase (DERA) has been determined by Se-Met multiple anomalous dispersion (MAD) methods at 0.99A resolution. This structure represents the highest-resolution X-ray structure of an aldolase determined to date and enables a true atomic view of the enzyme. The crystal structure shows the ubiquitous TIM alpha/beta barrel fold. The enzyme contains two lysine residues in the active site. Lys167 forms the Schiff base intermediate, whereas Lys201, which is in close vicinity to the reactive lysine residue, is responsible for the perturbed pK(a) of Lys167 and, hence, also a key residue in the reaction mechanism. DERA is the only known aldolase that is able to use aldehydes as both aldol donor and acceptor molecules in the aldol reaction and is, therefore, of particular interest as a biocatalyst in synthetic organic chemistry. The uncomplexed DERA structure enables a detailed comparison with the substrate complexes and highlights a conformational change in the phosphate-binding site. Knowledge of the enzyme active-site environment has been the basis for exploration of catalysis of non-natural substrates and of mutagenesis of the phosphate-binding site to expand substrate specificity. Detailed comparison with other class I aldolase enzymes and DERA enzymes from different organisms reveals a similar geometric arrangement of key residues and implies a potential role for water as a general base in the catalytic mechanism.     

Role of the yiaR and yiaS genes of Escherichia coli in metabolism of endogenously formed L-xylulose

Genes yiaP and yiaR of the yiaKLMNOPQRS cluster of Escherichia coli are required for the metabolism of the endogenously formed L-xylulose, whereas yiaS is required for this metabolism only in araD mutants. Like AraD, YiaS was shown to have L-ribulose-5-phosphate 4-epimerase activity. Similarity of YiaR to several 3-epimerases suggested that this protein could catalyze the conversion of L-xylulose-5-phosphate into L-ribulose-5-phosphate, thus completing the pathway between L-xylulose and the general metabolism.     

The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase

Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. In this study, the substrate preference of this enzyme was investigated by mutational analysis, X-ray crystallography and homology modelling. The crystal structure of hCBG was solved by the molecular replacement method and refined at 2.7 A resolution. The main-chain fold of the enzyme belongs to the (beta/alpha)(8) barrel structure, which is common to family 1 glycoside hydrolases. The active site is located at the bottom of a pocket (about 16 A deep) formed by large surface loops, surrounding the C termini of the barrel of beta-strands. As for all the clan of GH-A enzymes, the two catalytic glutamate residues are located on strand 4 (the acid/base Glu165) and on strand 7 (the nucleophile Glu373). Although many features of hCBG were shown to be very similar to previously described enzymes from this family, crucial differences were observed in the surface loops surrounding the aglycone binding site, and these are likely to strongly influence the substrate specificity. The positioning of a substrate molecule (quercetin-4'-glucoside) by homology modelling revealed that hydrophobic interactions dominate the binding of the aglycone moiety. In particular, Val168, Trp345, Phe225, Phe179, Phe334 and Phe433 were identified as likely to be important in determining substrate specificity in hCBG, and site-directed mutagenesis supported a key role for some of these residues.     

Structural basis for the function of pyridoxine 5'-phosphate synthase

BACKGROUND: Pyridoxal 5'-phosphate is the active form of vitamin B(6) that acts as an essential, ubiquitous coenzyme in amino acid metabolism. In Escherichia coli, the pathway of the de novo biosynthesis of vitamin B(6) results in the formation of pyridoxine 5'-phosphate (PNP), which can be regarded as the first synthesized B(6) vitamer. PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pathway, a complex intramolecular condensation reaction between 1-deoxy-D-xylulose-5'-phosphate and 1-amino-acetone-3-phosphate. RESULTS: The crystal structure of E. coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolution of 2.0 A. The monomer of PNP synthase consists of one compact domain that adopts the abundant TIM barrel fold. Intersubunit contacts are mediated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 symmetry. In the shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer. Furthermore, the structure of PNP synthase with its physiological products, PNP and P(i), was determined at 2.3 A resolution, which provides insight into the dynamic action of the enzyme and allows us to identify amino acids critical for enzymatic function. CONCLUSION: The high-resolution structures of the free enzyme and the enzyme-product complex of E. coli PNP synthase suggest essentials of the enzymatic mechanism. The main catalytic features are active site closure upon substrate binding by rearrangement of one C-terminal loop of the TIM barrel, charge-charge stabilization of the protonated Schiff-base intermediate, the presence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed state. All related PNP synthases are predicted to fold into a similar TIM barrel pattern and have comparable active site architecture. Thus, a common mechanism can be anticipated.     

N-acetylmannosamine-6-phosphate 2-epimerase uses a novel substrate-assisted mechanism to catalyze amino sugar epimerization

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation–reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.     

Crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, a crucial enzyme in the non-mevalonate pathway of isoprenoid biosynthesis.

We have solved the 2.5-A crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp(150), Glu(152), Glu(231), and Glu(234) are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed.     

Clusters in alpha/beta barrel proteins: implications for protein structure, function, and folding: a graph theoretical approach

The alpha/beta barrel fold is adopted by most enzymes performing a variety of catalytic reactions, but with very low sequence similarity. In order to understand the stabilizing interactions important in maintaining the alpha/beta barrel fold, we have identified residue clusters in a dataset of 36 alpha/beta barrel proteins that have less than 10% sequence identity within themselves. A graph theoretical algorithm is used to identify backbone clusters. This approach uses the global information of the nonbonded interaction in the alpha/beta barrel fold for the clustering procedure. The nonbonded interactions are represented mathematically in the form of an adjacency matrix. On diagonalizing the adjacency matrix, clusters and cluster centers are obtained from the highest eigenvalue and its corresponding vector components. Residue clusters are identified in the strand regions forming the beta barrel and are topologically conserved in all 36 proteins studied. The residues forming the cluster in each of the alpha/beta protein are also conserved among the sequences belonging to the same family. The cluster centers are found to occur in the middle of the strands or in the C-terminal of the strands. In most cases, the residues forming the clusters are part of the active site or are located close to the active site. The folding nucleus of the alpha/beta fold is predicted based on hydrophobicity index evaluation of residues and identification of cluster centers. The predicted nucleation sites are found to occur mostly in the middle of the strands. Proteins 2001;43:103-112.     

The structure of UDP-N-acetylglucosamine 2-epimerase reveals homology to phosphoglycosyl transferases

Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 A) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage beta-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar alpha/beta/alpha sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10 degrees interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc "hydrolyzing" 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.