Probing the Ser-Ser-Lys catalytic triad mechanism of peptide amidase: computational studies of the ground state, transition state, and intermediate

Peptide amidase (Pam), a hydrolytic enzyme that belongs to the amidase signature (AS) family, selectively catalyzes the hydrolysis of the C-terminal amide bond (CO-NH(2)) of peptides. The recent availability of the X-ray structures of Pam, fatty acid amide hydrolase, and malonamidase E2 has led to the proposal of a novel Ser-Ser-Lys catalytic triad mechanism for the amide hydrolysis by the AS enzymes. The molecular dynamics (MD) simulations using the CHARMM force field were performed to explore the catalytic mechanism of Pam. The 1.8 A X-ray crystal structure of Pam in complex with the amide analogue of chymostatin was chosen for the initial coordinates for the MD simulations. The five systems that were investigated are as follows: (i) enzyme.substrate with Lys123-NH(2), (ii) enzyme.substrate with Lys123-NH(3)(+), (iii) enzyme.substrate with Lys123-NH(3)(+) and Ser226-O(-), (iv) enzyme.transition state, and (v) enzyme.tetrahedral intermediate. Our data support the presence of the hydrogen bonding network among the catalytic triad residues, Ser226, Ser202, and Lys123, where Ser226 acts as the nucleophile and Ser202 bridges Ser226 and Lys123. The MD simulation supports the catalytic role of the crystallographic waters, Wat1 and Wat2. In all the systems that have been studied, the backbone amide nitrogens of Asp224 and Thr223 create an oxyanion hole by hydrogen bonding to the terminal amide oxygen of the substrate, and stabilize the oxyanion tetrahedral intermediate. The results from both our computational investigation and previously published experimental pH profile support two mechanisms. In a mechanism that is relevant at lower pH, the Lys123-NH(3)(+)-Ser202 dyad provides structural support to the catalytic residue Ser226, which in turn carries out a nucleophilic attack at the substrate amide carbonyl in concert with Wat1-mediated deprotonation and stabilization of the tetrahedral transition state by the oxyanion hole. In the mechanism operating at higher pH, the Lys123-NH(2)-Ser202 catalytic dyad acts as a general base to assist addition of Ser226 to the substrate amide carbonyl. The results from the MD simulation of the tetrahedral intermediate state show that both Ser202 and Lys123 are possible candidates for protonation of the leaving group, NH(2), to form the acyl-enzyme intermediate.     

The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.     

A trimetal site and substrate distortion in a family II inorganic pyrophosphatase

We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. Structures of both the fluoride-inhibited wild type and the H98Q variant of the PNP-Bacillus subtilis pyrophosphatase complex show a unique trinuclear metal center. Each metal ion coordinates a terminal oxygen on the electrophilic phosphate and a lone pair on the putative nucleophile, thus placing it in line with the scissile bond without any coordination by protein. The nucleophile moves further away from the electrophilic phosphorus site, to the opposite side of the trimetal plane, upon binding of substrate. In comparison with earlier product complexes, the side chain of Lys296 has swung in and so three positively charged side chains, His98, Lys205 and Lys296, now surround the bridging nitrogen in PNP. Finally, one of the active sites in the wild-type structure appears to show evidence of substrate distortion. Binding to the enzyme may thus strain the substrate and thus enhance the catalytic rate.     

Identification of catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by gamma-monofluorophosphono derivative of glutamic acid: N-terminal thr-391 in small subunit is the nucleophile

gamma-Glutamyltranspeptidase (EC 2.3.2.2) is the enzyme involved in glutathione metabolism and catalyzes the hydrolysis and transpeptidation of gamma-glutamyl compounds such as glutathione and its derivatives. The reaction is thought to proceed via a gamma-glutamyl-enzyme intermediate where a hitherto unknown catalytic nucleophile is gamma-glutamylated. Neither affinity labeling nor site-directed mutagenesis of conserved amino acids has succeeded so far in identifying the catalytic nucleophile. We describe here the identification of the catalytic nucleophile of Escherichia coli gamma-glutamyltranspeptidase by a novel mechanism-based affinity labeling agent, 2-amino-4-(fluorophosphono)butanoic acid (1), a gamma-phosphonic acid monofluoride derivative of glutamic acid. Compound 1 rapidly inactivated the enzyme in a time-dependent manner (k(on) = 4.83 x 10(4) M(-1) s(-1)). The inactivation rate was decreased by increasing the concentration of the substrate. The inactivated enzyme did not regain its activity after prolonged dialysis, suggesting that 1 served as an active-site-directed affinity label by phosphonylating the putative catalytic nucleophile. Ion-spray mass spectrometric analyses revealed that one molecule of 1 phosphonylated one molecule of the small subunit. LC/MS experiments of the proteolytic digests of the phosphonylated small subunit identified the N-terminal peptide Thr391-Lys399 as the phosphonylation site. Subsequent MS/MS experiments of this peptide revealed that the phosphonylated residue was Thr-391, the N-terminal residue of the small subunit. We conclude that the N-terminal Thr-391 is the catalytic nucleophile of E. coli gamma-glutamyltranspeptidase. This result strongly suggests that gamma-glutamyltranspeptidase is a new member of the N-terminal nucleophile hydrolase family.     

Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase.

The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom. The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175). In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups. The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176). The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8).     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.     

Structural and functional importance of first-shell metal ligands in the binuclear manganese cluster of arginase I

Arginase is a binuclear manganese metalloenzyme that hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of D128E, D128N, D232A, D232C, D234E, H101N, and H101E arginases I have been determined by X-ray crystallographic methods to elucidate the roles of the first-shell metal ligands in the stability and catalytic activity of the enzyme. This work represents the first structure-based dissection of the binuclear manganese cluster using site-directed mutagenesis and X-ray crystallography. Substitution of the metal ligands compromises the catalytic activity of the enzyme, either by the loss or disruption of the metal cluster or the nucleophilic metal-bridging hydroxide ion. However, the substitution of the metal ligands or the reduction of Mn(2+)(A) or Mn(2+)(B) occupancy does not compromise enzyme-substrate affinity as reflected by K(M), which remains relatively invariant across this series of arginase variants. This implicates a nonmetal binding site for substrate l-arginine in the precatalytic Michaelis complex, as proposed based on analysis of the native enzyme structure (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W. (1996) Nature 383, 554-557).     

A role for carbon catabolite repression in the metabolism of phosphonoacetate by Agromyces fucosus Vs2

A strain of Agromyces fucosus, designated Vs2, metabolized a range of organophosphonate compounds as sole phosphorus sources for growth and metabolized phosphonoacetate as a sole carbon, energy and phosphorus source for growth. With phosphonoacetate as the sole phosphorus source and a pyruvate carbon source, transient phosphate release to the medium was observed, in contrast to cultures grown with glucose and phosphonoacetate, where no phosphate release to the medium was observed. Carbon catabolite repression, specifically by means of inducer exclusion of phosphonoacetate, was proposed as the mechanism responsible, and phosphonoacetate hydrolase enzyme assays carried out on cell extracts confirmed that induced phosphonoacetate hydrolase activities were indeed higher in cells grown on pyruvate with phosphonoacetate as sole phosphorus source. This phenomenon has not previously been demonstrated in vivo, and must represent a significant metabolic control of organophosphonate metabolism. The catabolite repression phenomenon was also evident when A. fucosus grew on 2-aminoethylphosphonate as sole phosphorus source, allowing demonstration of a third mode of control for biodegradation of this compound. Excision of stained zymogram gel pieces, followed by tryptic digestion and mass spectrometric analysis, allowed the identification of phosphonoacetate hydrolase-derived peptides.     

Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type

T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β‐1,4‐glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09‐Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated N?2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water‐binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high‐resolution X‐ray structure of T26H at 1.04‐Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.     

Mechanism of the dihydroorotase reaction

Dihydroorotase (DHO) is a zinc metalloenzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. A chemical mechanism was proposed on the basis of an analysis of the effects of pH, metal substitution, solvent isotope effects, mutant proteins, and alternative substrates on the enzyme-catalyzed reaction. The pH-rate profiles for the hydrolysis of dihydroorotate or thiodihydroorotate demonstrated that a single group from the enzyme must be unprotonated for maximal catalytic activity. Conversely, the pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or cadmium by reconstitution of the apoenzyme with divalent cations in the presence of bicarbonate. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of the residue (Asp-250) that hydrogen bonds to the bridging hydroxide (or water) resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp-250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during hydrolysis of dihydroorotate. The X-ray crystal structure of DHO showed that the exocyclic alpha-carboxylate of dihydroorotate is bound to the protein via electrostatic interactions with Arg-20, Asn-44, and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thio analogue of dihydroorotate was found to be a good substrate of the enzyme. A comprehensive chemical mechanism for DHO was proposed on the basis of the experimental findings in this study and the X-ray crystal structure.     

Catalysis by nucleoside hydrolases

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Because of their vital role in the protozoan purine salvage pathway, nucleoside hydrolases from parasitic protozoa in particular have been studied extensively by X-ray crystallography, kinetic methods and site-directed mutagenesis. An elaborate network of conserved interactions between the metalloenzyme and the ribose enables steric and electrostatic stabilisation of the oxocarbenium-ion-like transition state. Activation of the leaving group by protonation before the formation of the transition state is a recurring catalytic strategy of enzymes that cleave N-glycosidic bonds. However, the mechanisms underlying leaving group activation are still the subject of debate for the nucleoside hydrolases.     

Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.     

Evidence for distinct roles in catalysis for residues of the serine-serine-lysine catalytic triad of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a mammalian amidase signature enzyme that inactivates neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The recent determination of the three-dimensional structures of FAAH and two distantly related bacterial amidase signature enzymes indicates that these enzymes employ an unusual serine-serine-lysine triad for catalysis (Ser-241/Ser-217/Lys-142 in FAAH). Mutagenesis of each of the triad residues in FAAH has been shown to severely reduce amidase activity; however, how these residues contribute, both individually and in cooperation, to catalysis remains unclear. Here, through a combination of site-directed mutagenesis, enzyme kinetics, and chemical labeling experiments, we provide evidence that each FAAH triad residue plays a distinct role in catalysis. In particular, the mutation of Lys-142 to alanine indicates that this residue functions as both a base involved in the activation of the Ser-241 nucleophile and an acid that participates in the protonation of the substrate leaving group. This latter property appears to support the unusual ability of FAAH to hydrolyze amides and esters at equivalent rates. Interestingly, although structural evidence indicates that the impact of Lys-142 on catalysis probably occurs through the bridging Ser-217, the mutation of this latter residue to alanine impaired catalytic activity but left the amide/ester hydrolysis ratios of FAAH intact. Collectively, these findings suggest that FAAH possesses a specialized active site structure dedicated to a mechanism for competitive amide and ester hydrolysis where nucleophile attack and leaving group protonation occur in a coordinated manner dependent on Lys-142.     

A comparison of three bacterial phosphonoacetate hydrolases from different environmental sources

Cleavage of the carbon–phosphorus bond of the xenobiotic phosphonoacetate by phosphonoacetate hydrolase represents a novel route for the microbial metabolism of organophosphonates, and is unique in that it is substrate-inducible and its expression is independent of the phosphate status of the cell. The enzyme has previously only been demonstrated in cell extracts of Pseudomonas fluorescens 23F. Phosphonoacetate hydrolase activity is now reported in extracts of environmental Curtobacterium sp. and Pseudomonas sp. isolates capable of the phosphate-insensitive mineralization of phosphonoacetate as the sole source of carbon, energy and phosphorus at concentrations up to 40 mmol l⁻¹ and 100 mmol l⁻¹, respectively. The enzymes in both strains were similarly inducible by phosphonoacetate and had a unique specificity for this substrate. However, they differed significantly from each other, and from the previously described Ps. fluorescens 23F enzyme, in respect of their apparent molecular masses, temperature optima, thermostability, sensitivity to inhibition by chelating agents and by structural analogues of phosphonoacetate, and in their affinities for the substrate.     

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-beta-D-xylobioside substrate was increased by 10(3)-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the alpha-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.     

The crystal structures of phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme's catalytic mechanism.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in the coenzyme A pathway that catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) in the presence of magnesium. To investigate the reaction mechanism, the high-resolution crystal structures of the Escherichia coli PPAT have been determined in the presence of either ATP or Ppant. Structural details of the catalytic center revealed specific roles for individual amino acid residues involved in substrate binding and catalysis. The side-chain of His18 stabilizes the expected pentacovalent intermediate, whereas the side-chains of Thr10 and Lys42 orient the nucleophile for an in-line displacement mechanism. The binding site for the manganese ion that interacts with the phosphate groups of the nucleotide has also been identified. Within the PPAT hexamer, one trimer is in its substrate-free state, whereas the other is in a substrate-bound state.     

Mechanism and diversity of the erythromycin esterase family of enzymes

Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.     

Significant enhancement in the binding of p-nitrophenyl-β- -xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E-86

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-β- -xylobioside substrate was increased by 10³-fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the α-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.     

Catalytic mechanism of class B2 metallo-beta-lactamase

The initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-beta-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be approximately 14 kcal/mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the O(delta2)-Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-beta-lactamase.     

Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process.