Peptidoglycan amidase MepA is a LAS metallopeptidase

LAS enzymes are a group of metallopeptidases that share an active site architecture and a core folding motif and have been named according to the group members lysostaphin, D-Ala-D-Ala carboxypeptidase and sonic hedgehog. Escherichia coli MepA is a periplasmic, penicillin-insensitive murein endopeptidase that cleaves the D-alanyl-meso-2,6-diamino-pimelyl amide bond in E. coli peptidoglycan. The enzyme lacks sequence similarity with other peptidases, and is currently classified as a peptidase of unknown fold and catalytic class in all major data bases. Here, we build on our observation that two motifs, characteristic of the newly described LAS group of metallopeptidases, are conserved in MepA-type sequences. We demonstrate that recombinant E. coli MepA is sensitive to metal chelators and that mutations in the predicted Zn2+ ligands His-113, Asp-120, and His-211 inactivate the enzyme. Moreover, we present the crystal structure of MepA. The active site of the enzyme is most similar to the active sites of lysostaphin and D-Ala-D-Ala carboxypeptidase, and the fold is most closely related to the N-domain of sonic hedgehog. We conclude that MepA-type peptidases are LAS enzymes.     

Similar active sites in lysostaphins and D-Ala-D-Ala metallopeptidases

Specific peptidases exist for nearly every amide linkage in peptidoglycan. In several cases, families of peptidoglycan hydrolases with different specificities turned out to be related. Here we show that lysostaphin-type peptidases and D-Ala-D-Ala metallopeptidases have similar active sites and share a core folding motif in otherwise highly divergent folds. The central Zn(2+) is tetrahedrally coordinated by two histidines, an aspartate, and a water molecule. The Zn(2+) chelating residues occur in the order histidine, aspartate, histidine in all sequences and contact the metal via the Nepsilon, the Odelta, and the Ndelta, respectively. The identity of the other active-site residues varies, but in all enzymes of known structure except for VanX, a conserved histidine is present two residues upstream of the second histidine ligand to the Zn(2+). As the same arrangement of active-site residues is also found in the N-terminal, cryptic peptidase domain of sonic hedgehog, we propose that this arrangement of active-site residues be called the "LAS" arrangement, because it is present in lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and in the cryptic peptidase in the N-domain of sonic hedgehog.     

Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.     

Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

Glutamic acid residues as metal ligands in the active site of Escherichia coli alkaline phosphatase

Four independent mutations were introduced to the Escherichia coli alkaline phosphatase active site, and the resulting enzymes characterized to study the effects of Glu as a metal ligand. The mutations D51E and D153E were created to study the effects of lengthening the carboxyl group by one methylene unit at the metal interaction site. The D51E enzyme had drastically reduced activity and lost one zinc per active site, demonstrating importance of the position of Asp(51). The D153E enzyme had an increased k(cat) in the presence of high concentrations of Mg(2+), along with a decreased Mg(2+) affinity as compared to the wild-type enzyme. The H331E and H412E enzymes were created to probe the requirement for a nitrogen-containing metal ligand at the Zn(1) site. The H331E enzyme had greatly decreased activity, and lost one zinc per active site. In the absence of high concentrations of Zn(2+), dephosphorylation occurs at an extremely reduced rate for the H412E enzyme, and like the H331E enzyme, metal affinity is reduced. Except at the 153 position, Glu is not an acceptable metal chelating amino acid at these positions in the E. coli alkaline phosphatase active site.     

Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle.

Tyrosinase (Tyr) and tyrosinase-related proteins (Tyrps) 1 and 2 are the enzymes responsible for mammalian melanogenesis. They display high similarity but different substrate and reaction specificities. Loss-of-function mutations lead to several forms of albinism or other pigmentation disorders. They share two conserved metal binding sites (CuA and CuB) which, in Tyr, bind copper. To define some structural determinants for these differences, we mutated Tyr at selected residues on the basis of (i) conservation of the original residues in most tyrosinases, (ii) their nonconservative substitution in the Tyrps, and (iii) their possible involvement as an endogenous bridge between the copper pair. Two mutations at the CuA site, S192A and E193Q, did not affect Tyr activities, thus excluding S192 and E193 as endogenous ligands of the copper pair. Concerning CuB, the H390Q mutation completely abolished Tyr activity, whereas Q378H and H389L mutants showed 10-20% residual specific activities. Their kinetic behavior suggests that (i) H390 is the actual third ligand for CuB, (ii) H389 is critical for stereospecific recognition of o-diphenols but not monophenols, and (iii) the involvement in metal binding of the central extra H residue at the Tyrps CuB site is unlikely. However, replacement of Q (in Tyr) by H (in Tyrps) greatly diminished the affinity for L-dopa, consistent with the low/null tyrosinase activity of the Tyrps. These are the first data showing a physical difference in docking of mono- and o-diphenols to the Tyr active site, and they are used to propose a revised scheme of the catalytic cycle.     

Identification of iron ligands in tyrosine hydroxylase by mutagenesis of conserved histidinyl residues.

Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine and other aromatic amino acids using a tetrahydropterin as the reducing substrate. The enzyme is a homotetramer; each monomer contains a single nonheme iron atom. Five histidine residues are conserved in all tyrosine hydroxylases that have been sequenced to date and in the related eukaryotic enzymes phenylalanine and tryptophan hydroxylase. Because histidine has been suggested as a ligand to the iron in these enzymes, mutant tyrosine hydroxylase proteins in which each of the conserved histidines had been mutated to glutamine or alanine were expressed in Escherichia coli. The H192Q, H247Q, and H317A mutant proteins contained iron in comparable amounts to the wild-type enzyme, about 0.6 atoms/sub-unit. In contrast, the H331 and H336 mutant proteins contained no iron. The first three mutant enzymes were active, with Vmax values 39, 68, and 7% that of the wild-type enzyme, and slightly altered V/Km values for both tyrosine and 6-methyltetrahydropterin. In contrast, the H331 and H336 mutant enzymes had no detectable activity. The EPR spectra of the H192Q and H247Q enzymes are indistinguishable from that of wild-type tyrosine hydroxylase, whereas that of the H317A enzyme indicated that the ligand field of the iron had been slightly perturbed. These results are consistent with H331 and H336 being ligands to the active site iron atom.     

Characterization of metal ligand mutants of tyrosine hydroxylase: insights into the plasticity of a 2-histidine-1-carboxylate triad

The amino acid ligands to the active site iron in the aromatic amino acid hydroxylase tyrosine hydroxylase are two histidines and a glutamate. This 2-histidine-1-carboxylate motif has been found in a number of other metalloenzymes which catalyze a variety of oxygenase reactions. As a probe of the plasticity of this metal binding site, each of the ligands in TyrH has been mutated to glutamine, glutamate, or histidine. The H336E and H336Q enzymes show dramatic decreases in iron affinity but retain substantial activity for both tyrosine hydroxylation and tetrahydropterin oxidation. The H331E enzyme shows a lesser decrease in iron affinity and is unable to hydroxylate tyrosine. Instead, this enzyme oxidizes tetrahydropterin in the absence of added tyrosine. The E376H enzyme has no significant activity, while the E376Q enzyme hydroxylates tyrosine at about 0.4% the wild-type rate. When dopamine is bound to either the H336Q or H331E enzymes, the position of the long wavelength charge-transfer absorbance band is consistent with the change in the metal ligand. In contrast, the H336E enzyme does not form a stable binary complex with dopamine, while the E376H and E376Q enzymes catalyze dopamine oxidation.     

Structural characterization of a putative endogenous metal chelator in the periplasmic nickel transporter NikA

Escherichia coli and related bacteria require nickel for the synthesis of hydrogenases, enzymes involved in hydrogen oxidation and proton reduction. Nickel transport to the cytoplasm depends on five proteins, NikA-E. We have previously reported the three-dimensional structure of the soluble periplasmic nickel transporter NikA in a complex with FeEDTA(H 2O) (-). We have now determined the structure of EDTA-free NikA and have found that it binds a small organic molecule that contributes three ligands to the coordination of a transition metal ion. Unexpectedly, His416, which was far from the metal-binding site in the FeEDTA(H 2O) (-)-NikA complex, becomes the fourth observed ligand to the metal. The best match to the omit map electron density is obtained for butane-1,2,4-tricarboxylate (BTC). Our attempts to obtain a BTC-Ni-NikA complex using apo protein and commercial reagents resulted in nickel-free BTC-NikA. Overall, our results suggest that nickel transport in vivo requires a specific metallophore that may be BTC.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Crystal structure of Escherichia coli PdxA,an enzyme involved in the pyridoxal phosphate biosynthesis pathway

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn2+. The protein forms tightly bound dimers. Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.     

An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes.     

EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC)

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.     

Zinc coordination sphere in biochemical zinc sites

Zinc is known to be indispensable to growth and development and transmission of the genetic message. It does this through a remarkable mosaic of zinc binding motifs that orchestrate all aspects of metabolism. There are now nearly 200 three dimensional structures for zinc proteins, representing all six classes of enzymes and covering a wide range of phyla and species. These structures provide standards of reference for the identity and nature of zinc ligands in other proteins for which only the primary structure is known. Three primary types of zinc sites are apparent from examination of these structures: structural, catalytic and cocatalytic. The most common amino acids that supply ligands to these sites are His, Glu, Asp and Cys. In catalytic sites zinc generally forms complexes with water and any three nitrogen, oxygen and sulfur donors with His being the predominant amino acid chosen. Water is always a ligand to such sites. Structural zinc sites have four protein ligands and no bound water molecule. Cys is the preferred ligand in such sites. Cocatalytic sites contain two or three metals in close proximity with two of the metals bridged by a side chain moiety of a single amino acid residue, such as Asp, Glu or His and sometimes a water molecule. Asp and His are the preferred amino acids for these sites. No Cys ligands are found in such sites. The scaffolding of the zinc sites is also important to the function and reactivity of the bound metal. The influence of zinc on quaternary protein structure has led to the identification of a fourth type of zinc binding site, protein inteface. In this case zinc sites are formed from ligands supplied from amino acid residues residing in the binding surface of two proteins. The resulting zinc site usually has the coordination properties of a catalytic or structural zinc binding site.     

An XAS investigation of product and inhibitor complexes of Ni-containing GlxI from Escherichia coli: mechanistic implications

Escherichia coli glyoxalase I (GlxI) is a metalloisomerase that is maximally activated by Ni(2+), unlike other known GlxI enzymes which are active with Zn(2+). The metal is coordinated by two aqua ligands, two histidines (5 and 74), and two glutamates (56 and 122). The mechanism of E. coli Ni-GlxI was investigated by analyzing Ni K-edge X-ray absorption spectroscopic (XAS) data obtained from the enzyme and complexes formed with the product, S-D-lactoylglutathione, and various inhibitors. The analysis of X-ray absorption near edge structure (XANES) was used to determine the coordination number and geometry of the Ni site in the various Ni-GlxI complexes. Metric details of the Ni site structure were obtained from the analysis of extended X-ray absorption fine structure (EXAFS). Interaction of S-D-lactoylglutathione (product) or octylglutathione with the enzyme did not change the structure of the Ni site. However, analysis of XAS data obtained from a complex formed with a peptide hydroxamate bound to Ni-GlxI is consistent with this inhibitor binding to the Ni center by displacement of both water molecules. XANES analysis of this complex is best fit with a five-coordinate metal and, given the fact that both histidine ligands are retained, suggests the loss of a glutamate ligand. The loss of a glutamate ligand would preserve the neutral charge on the Ni complex and is consistent with the lack of a significant shift in the Ni K-edge energy in this complex. These data are compared with data obtained from the E. coli Ni-GlxI selenomethionine-substituted enzyme. The replacement of three methionine residues in the native enzyme with selenomethionine does not affect the structure of the Ni site. However, addition of the peptide hydroxamate inhibitor leads to the formation of a complex whose structure as determined by XAS analysis is consistent with inhibitor binding via displacement of both water molecules but retention of both histidine and glutamate ligands. This leads to an anionic complex, which is consistent with an observed 1.7 eV decrease in the Ni K-edge energy. Plausible reaction mechanisms for Ni-GlxI are discussed in light of the structural information available.     

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding

We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.     

Similarity-driven flexible ligand docking

A similarity-driven approach to flexible ligand docking is presented. Given a reference ligand or a pharmacophore positioned in the protein active site, the method allows inclusion of a similarity term during docking. Two different algorithms have been implemented, namely, a similarity-penalized docking (SP-DOCK) and a similarity-guided docking (SG-DOCK). The basic idea is to maximally exploit the structural information about the ligand binding mode present in cases where ligand-bound protein structures are available, information that is usually ignored in standard docking procedures. SP-DOCK and SG-DOCK have been derived as modified versions of the program DOCK 4.0, where the similarity program MIMIC acts as a module for the calculation of similarity indices that correct docking energy scores at certain steps of the calculation. SP-DOCK applies similarity corrections to the set of ligand orientations at the end of the ligand incremental construction process, penalizing the docking energy and, thus, having only an effect on the relative ordering of the final solutions. SG-DOCK applies similarity corrections throughout the entire ligand incremental construction process, thus affecting not only the relative ordering of solutions but also actively guiding the ligand docking. The performance of SP-DOCK and SG-DOCK for binding mode assessment and molecular database screening is discussed. When applied to a set of 32 thrombin ligands for which crystal structures are available, SG-DOCK improves the average RMSD by ca. 1 A when compared with DOCK. When those 32 thrombin ligands are included into a set of 1,000 diverse molecules from the ACD, DIV, and WDI databases, SP-DOCK significantly improves the retrieval of thrombin ligands within the first 10% of each of the three databases with respect to DOCK, with minimal additional computational cost. In all cases, comparison of SP-DOCK and SG-DOCK results with those obtained by DOCK and MIMIC is performed.     

The electrostatic potential of Escherichia coli dihydrofolate reductase

Escherichia coli dihydrofolate reductase (DHFR) carries a net charge of -10 electrons yet it binds ligands with net charges of -4 (NADPH) and -2 (folate or dihydrofolate). Evaluation and analysis of the electrostatic potential of the enzyme give insight as to how this is accomplished. The results show that the enzyme is covered by an overall negative potential (as expected) except for the ligand binding sites, which are located inside "pockets" of positive potential that enable the enzyme to bind the negatively charged ligands. The electrostatic potential can be related to the asymmetric distribution of charged residues in the enzyme. The asymmetric charge distribution, along with the dielectric boundary that occurs at the solvent-protein interface, is analogous to the situation occurring in superoxide dismutase. Thus DHFR is another case where the shape of the active site focuses electric fields out into solution. The positive electrostatic potential at the entrance of the ligand binding site in E. coli DHFR is shown to be a direct consequence of the presence of three positively charged residues at positions 32, 52, and 57--residues which have also been shown recently to contribute significantly to electronic polarization of the ligand folate. The latter has been postulated to be involved in the catalytic process. A similar structural motif of three positively charged amino acids that gives rise to a positive potential at the entrance to the active site is also found in DHFR from chicken liver, and is suggested to be a common feature in DHFRs from many species. It is noted that, although the net charges of DHFRs from different species vary from +3 to -10, the enzymes are able to bind the same negatively charged ligands, and perform the same catalytic function.     

Crystal structure of the PdxY Protein from Escherichia coli

The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-A resolution. PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5' hydroxyl. The protein is a homodimer with an active site on each monomer composed of residues that come exclusively from each respective subunit. The active site is filled with a density that fits that of pyridoxal. In monomer A, the ligand appears to be covalently attached to Cys122 as a thiohemiacetal, while in monomer B it is not covalently attached but appears to be partially present as pyridoxal 5'-phosphate. The presence of pyridoxal phosphate and pyridoxal as ligands was confirmed by the activation of aposerine hydroxymethyltransferase after release of the ligand by the denaturation of PdxY. The ligand, which appears to be covalently attached to Cys122, does not dissociate after denaturation of the protein. A detailed comparison (of functional properties, sequence homology, active site and ATP-binding-site residues, and active site flap types) of PdxY with other pyridoxal kinases as well as the ribokinase superfamily in general suggested that PdxY is a member of a new subclass of the ribokinase superfamily. The structure of PdxY also permitted an interpretation of work that was previously published about this enzyme.