Acyl-intermediate structures of the extended-spectrum class A beta-lactamase,Toho-1, in complex with cefotaxime,cephalothin, and benzylpenicillin

Bacterial resistance to beta-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of beta-lactamases that inactivate beta-lactam antibiotics. Toho-1 is an extended-spectrum beta-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former beta-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-A resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the Omega-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn(104) and Asp(240) form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser(237) and cephalosporins probably helps to bring the beta-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.     

Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor

Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo beta-lactamases. Reported here are the crystal structures of the native IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4 -(phenylb utyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 A (native) and 2.0 A (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo beta-lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 A compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the beta-lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo beta-lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo beta-lactamase inhibition with utility for future drug design.     

Substitution of lysine at position 104 or 240 of TEM-1pTZ18R beta-lactamase enhances the effect of serine-164 substitution on hydrolysis or affinity for cephalosporins and the monobactam aztreonam

By site-directed mutagenesis, TEM-1 beta-lactamase was altered to contain single amino acid changes of E104K, R164S, and E240K, in addition to double changes of E104K/R164S or R164S/E240K and the triple change of E104K/R164S/E240K. Hydrolysis rates for cephaloridine and benzylpenicillin were lowered at least 1 order of magnitude for all enzymes containing R164S substitutions. All mutant enzymes exhibited increased kcat values for beta-lactam antibiotics containing an aminothiazole oxime side chain. Hydrolysis of ceftazidime was most affected, with kcat values increased 3-4 orders of magnitude in all enzymes with the substituted R164S moiety. Km values decreased for all substrates except ceftazidime in the enzymes with multiple mutations. Aztreonam was most affected, with Km values lowered 23-56-fold in the enzymes bearing multiple mutations. When the crystal structures of aztreonam and related monobactams were studied and projected into an active-site model of the PC1 beta-lactamase, it became apparent that the two lysine residues might serve equivalent roles by interacting with the carboxylate of the aminothiazole oxime side chain. Hydrogen-bonding interactions involving the oxime and N7 of the lysine, particularly Lys-104, may also be important in some antibiotics. Ser-164 apparently serves an indirect role, since it is somewhat distant from the active-site cleft.     

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.     

Enzyme-induced strain/distortion in the ground-state ES complex in beta-lactamase catalysis revealed by FTIR

Class A beta-lactamases hydrolyze penicillins and other beta-lactams via an acyl-enzyme catalytic mechanism. Ser70 is the active site nucleophile. By constructing the S70A mutant, which is unable to form the acyl-enzyme intermediate, it was possible to make stable ES complexes with various substrates. The stability of such Michaelis complexes permitted acquisition of their infrared spectra. Comparison of the beta-lactam carbonyl stretch frequency (nu(CO)) in the free and enzyme-bound substrate revealed an average decrease of 13 cm(-)(1), indicating substantial strain/distortion of the lactam carbonyl when bound in the ES complex. Interestingly, regardless of the frequency of the C=O stretch in the free substrate, when complexed to Bacillus licheniformis beta-lactamase, the frequency was always 1755 +/- 2 cm(-)(1). This suggests the active site environment induces a similar conformation of the beta-lactam in all substrates when bound to the enzyme. Using deuterium substitution, it was shown that the "oxyanion hole", which involves hydrogen bonding to two backbone amides, is the major source of the enzyme-induced strain/distortion. The very weak catalytic activity of the S70A beta-lactamase suggests enzyme-facilitated hydrolysis due to substrate distortion on binding to the enzyme. Thus the binding of the substrate in the active site induces substantial strain and distortion that contribute significantly to the overall rate enhancement in beta-lactamase catalysis.     

The activity of the dinuclear cobalt-beta-lactamase from Bacillus cereus in catalysing the hydrolysis of beta-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyse the hydrolysis of beta-lactam antibiotics, but are also able to function with cobalt(II) and require one or two metal-ions for catalytic activity. The hydrolysis of cefoxitin, cephaloridine and benzylpenicillin catalysed by CoBcII (cobalt-substituted beta-lactamase from Bacillus cereus) has been studied at different pHs and metal-ion concentrations. An enzyme group of pK(a) 6.52+/-0.1 is found to be required in its deprotonated form for metal-ion binding and catalysis. The species that results from the loss of one cobalt ion from the enzyme has no significant catalytic activity and is thought to be the mononuclear CoBcII. It appears that dinuclear CoBcII is the active form of the enzyme necessary for turnover, while the mononuclear CoBcII is only involved in substrate binding. The cobalt-substituted enzyme is a more efficient catalyst than the native enzyme for the hydrolysis of some beta-lactam antibiotics suggesting that the role of the metal-ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Relocation of the catalytic carboxylate group in class A beta-lactamase: the structure and function of the mutant enzyme Glu166-->Gln:Asn170-->Asp.

The hydrolysis of beta-lactam antibiotics by the serine-beta-lactamases proceeds via an acyl-enzyme intermediate. In the class A enzymes, a key catalytic residue, Glu166, activates a water molecule for nucleophilic attack on the acyl-enzyme intermediate. The active site architecture raises the possibility that the location of the catalytic carboxylate group may be shifted while still maintaining close proximity to the hydrolytic water molecule. A double mutant of the Staphylococcus aureus PC1 beta-lactamase, E166Q:N170D, was produced, with the carboxylate group shifted to position 170 of the polypeptide chain. A mutant protein, E166Q, without a carboxylate group and with abolished deacylation, was produced as a control. The kinetics of the two mutant proteins have been analyzed and the crystal structure of the double mutant protein has been determined. The kinetic data confirmed that deacylation was restored in E166Q:N170D beta-lactamase, albeit not to the level of the wild-type enzyme. In addition, the kinetics of the double mutant enzyme follows progressive inactivation, characterized by initial fast rates and final slower rates. The addition of ammonium sulfate increases the size of the initial burst, consistent with stabilization of the active form of the enzyme by salt. The crystal structure reveals that the overall fold of the E166Q:N170D enzyme is similar to that of native beta-lactamase. However, high crystallographic temperature factors are associated with the ohm-loop region and some of the side chains, including Asp170, are partially or completely disordered. The structure provides a rationale for the progressive inactivation of the Asp170-containing mutant, suggesting that the flexible ohm-loop may be readily perturbed by the substrate such that Asp170's carboxylate group is not always poised to facilitate hydrolysis.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.     

Crystal structure of gamma-chymotrypsin in complex with 7-hydroxycoumarin

The 1.8 A crystal structure of 7-hydroxycoumarin (7-HC) bound to chymotrypsin reveals that this inhibitor forms a planar cinnamate acyl-enzyme complex. The phenyl ring of the bound inhibitor forms numerous van der Waals contacts in the S1 pocket of the enzyme, with the p-hydroxyl group donating a hydrogen bond to the main-chain oxygen atom of Ser217, and the o-hydroxyl group forming a water-mediated hydrogen bond with the carbonyl oxygen of Val227. The structure of the acyl-enzyme complex suggests that the mechanism of inhibition of 7-HC involves nucleophilic attack by the Ser195 O(gamma) atom on the carbonyl carbon atom of the inhibitor, accompanied by the breaking of the 2-pyrone ring of the inhibitor, and leading to the formation of a cinnamate acyl-enzyme derivative via a tetrahedral transition state. Comparisons with structures of photoreversible cinnamates bound to chymotrypsin reveal that although 7-HC interacts with the enzyme in a similar fashion, the binding of 7-HC to chymotrypsin takes place in a productive conformation in contrast to the photoreversible cinnamates. In summary, the 7-HC-chymotrypsin complex provides basic insight into the inhibition of chymotrypsin by natural coumarins and provides a structural basis for the design of more potent mechanism-based inhibitors against a wide range of biologically important chymotrypsin-like enzymes.     

Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G beta-lactamase.

Residue Arg220 was found to be important for the acylation of the Streptomyces albus G beta-lactamase by classical penicillins and cephalosporins bearing a carboxylate on C3 or C4. The R220L mutant exhibited strongly decreased kcat/Km values for those compounds. Conversely the acylation rates by benzylpenicillin methylester and deacetylcephalosporin C lactone were little affected, indicating a direct or indirect role of that positively charged residue in the interaction of the enzyme cavity with the negative charge of the substrate. Surprisingly that residue is not conserved in all class A beta-lactamases but when it is not present it can be seen in the known tertiary structures that the guanidinium group of another arginine side chain (Arg244) is similarly positioned. The mutation affected the behaviour of the enzyme towards cephaloridine much less than towards cephalothin. This might represent an example of substrate-assisted catalysis where the disappearance of a positive charge on the enzyme is partly compensated by the presence of a similarly charged group on one of the substrate side chains. All the experimental results are nicely explained by computer-modelling of the enzyme-substrate interactions.     

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.     

Crystal structures of the class D beta-lactamase OXA-13 in the native form and in complex with meropenem.

The therapeutic problems posed by class D beta-lactamases, a family of serine enzymes that hydrolyse beta-lactam antibiotics following an acylation-deacylation mechanism, are increased by the very low level of sensitivity of these enzymes to beta-lactamase inhibitors. To gain structural and mechanistic insights to aid the design of new inhibitors, we have determined the crystal structure of OXA-13 from Pseudomonas aeruginosa in the apo form and in complex with the carbapenem meropenem. The native form consisted of a dimer displaying an overall organisation similar to that found in the closely related enzyme OXA-10. In the acyl-enzyme complex, the positioning of the antibiotic appeared to be ensured mainly by (i) the covalent acyl bond and (ii) a strong salt-bridge involving the carboxylate moiety of the drug. Comparison of the structures of OXA-13 in the apo form and in complex with meropenem revealed an unsuspected flexibility in the region of the essential serine 115 residue, with possible consequences for the catalytic properties of the enzyme. In the apo form, the Ser115 side-chain is oriented outside the active site, whereas the general base Lys70 adopts a conformation that seems to be incompatible with the activation of the catalytic water molecule required for the deacylation step. In the OXA-13:meropenem complex, a 3.5 A movement of the backbone of the 114-116 loop towards the side-chain of Lys70 was observed, which seems to be driven by a displacement of the neighbouring 91-104 loop and which results in the repositioning of the side-chain hydroxyl group of Ser115 toward the catalytic centre. Concomitantly, the side-chain of Lys70 is forced to curve in the direction of the deacylating water molecule, which is then strongly bound and activated by this residue. However, a distance of ca 5 A separates the catalytic water molecule from the acyl carbonyl group of meropenem, a structural feature that accounts for the inhibition of OXA-13 by this drug. Finally, the low level of penicillinase activity revealed by the kinetic analysis of OXA-13 could be related to the specific presence in position 73 of a serine residue located close to the general base Lys70, which results in a decrease of the number of hydrogen-bonding interactions stabilising the catalytic water molecule.     

Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin

Class A beta-lactamases are known to hydrolyze substrates through a Ser70-linked acyl-enzyme intermediate, although the detailed mechanism remains unknown. On the basis of the tertiary structure of the active site, the role of Glu166 of class A enzymes was investigated by replacing the residue in RTEM-1 beta-lactamase with Ala, Asp, Gln, or Asn. All the mutants, in contrast to the wild-type, accumulated a covalent complex with benzylpenicillin which corresponds to an acyl-enzyme intermediate. For the Asp mutant, the complex decayed slowly and the hydrolytic activity was slightly retained both in vivo and in vitro. In contrast, the other mutants lost the hydrolytic activity completely and their complexes were stable. These results indicate that the side-chain carboxylate of Glu166 acts as a special catalyst for deacylation. Residues for deacylation have not been identified in other acyl enzymes, such as serine proteases and class C beta-lactamases. Furthermore, the acyl-enzyme intermediates obtained are so stable that they are considered to be ideal materials for crystallographic studies for elucidating the catalytic mechanism in more detail. In addition, the mutants can more easily form inclusion bodies than the wild-type, when they are produced in a large amount, suggesting that the residue also plays an important role in proper folding of the enzyme.     

Inhibitor-resistant class A beta-lactamases: consequences of the Ser130-to-Gly mutation seen in Apo and tazobactam structures of the SHV-1 variant

A bacterial response to the clinical use of class A beta-lactamase inhibitors such as tazobactam and clavulanic acid is the expression of variant beta-lactamases with weaker binding affinities for these mechanism-based inhibitors. Some of these inhibitor-resistant variants contain a glycine mutation at Ser130, a conserved active site residue known to be adventitiously involved in the inhibition mechanism. The crystallographic structure of a complex of tazobactam with the Ser130Gly variant of the class A SHV-1 beta-lactamase has been determined to 1.8 A resolution. Two reaction intermediates are observed. The primary intermediate is an acyclic species bound to the reactive Ser70. It is poorly primed for catalytic hydrolysis because its ester carbonyl group is completely displaced from the enzyme's oxyanion hole. A smaller fraction of the enzyme contains a Ser70-bound aldehyde resulting from hydrolytic loss of the triazoyl-sulfinyl amino acid moiety from the primary species. This first structure of a class A beta-lactamase lacking Ser130, the side chain of which functions in beta-lactam binding and possibly in catalysis, gives crystallographic evidence that the acylation step of beta-lactam turnover can occur without Ser130. Unexpectedly, the crystal structure of the uncomplexed Ser130Gly enzyme, also determined to 1.8 A resolution, shows that a critical Glu166-activated water molecule is missing from the catalytic site. Comparison of this uncomplexed variant with the wild-type structure reveals that Ser130 is required for orienting the side chain of Ser70 and ensuring the hydrogen bonding of Ser70 to both Lys73 and the catalytic water molecule.     

Molecular dynamics simulations of the acyl-enzyme and the tetrahedral intermediate in the deacylation step of serine proteases

Despite the availability of many experimental data and some modeling studies, questions remain as to the precise mechanism of the serine proteases. Here we report molecular dynamics simulations on the acyl-enzyme complex and the tetrahedral intermediate during the deacylation step in elastase catalyzed hydrolysis of a simple peptide. The models are based on recent crystallographic data for an acyl-enzyme intermediate at pH 5 and a time-resolved study on the deacylation step. Simulations were carried out on the acyl enzyme complex with His-57 in protonated (as for the pH 5 crystallographic work) and deprotonated forms. In both cases, a water molecule that could provide the nucleophilic hydroxide ion to attack the ester carbonyl was located between the imidazole ring of His-57 and the carbonyl carbon, close to the hydrolytic position assigned in the crystal structure. In the "neutral pH" simulations of the acyl-enzyme complex, the hydrolytic water oxygen was hydrogen bonded to the imidazole ring and the side chain of Arg-61. Alternative stable locations for water in the active site were also observed. Movement of the His-57 side-chain from that observed in the crystal structure allowed more solvent waters to enter the active site, suggesting that an alternative hydrolytic process directly involving two water molecules may be possible. At the acyl-enzyme stage, the ester carbonyl was found to flip easily in and out of the oxyanion hole. In contrast, simulations on the tetrahedral intermediate showed no significant movement of His-57 and the ester carbonyl was constantly located in the oxyanion hole. A comparison between the simulated tetrahedral intermediate and a time-resolved crystallographic structure assigned as predominantly reflecting the tetrahedral intermediate suggests that the experimental structure may not precisely represent an optimal arrangement for catalysis in solution. Movement of loop residues 216-223 and P3 residue, seen both in the tetrahedral simulation and the experimental analysis, could be related to product release. Furthermore, an analysis of the geometric data obtained from the simulations and the pH 5 crystal structure of the acyl-enzyme suggests that since His-57 is protonated, in some aspects, this crystal structure resembles the tetrahedral intermediate.     

Kinetics and mechanism of the serine beta-lactamase catalyzed hydrolysis of depsipeptides

Steady-state kinetic parameters have been determined for the hydrolysis of a series of acyclic depsipeptides (ester analogues of acyl-D-alanyl-D-alanine peptides) catalyzed by representative class C (Enterobacter cloacae P99) and class A (Bacillus cereus I, TEM-2, and Staphylococcus aureus PC1) beta-lactamases. The best of these substrates, and the one most used in this work, was m-[[(phenylacetyl)-glycyl]oxy]benzoic acid, whose rates of cleavage could be followed spectrophotometrically. The P99 enzyme also catalyzed the methanolysis of these substrates in aqueous methanol solutions. Quantitative evaluation of the effects of methanol on the kinetics of the competing hydrolysis and methanolysis reactions, and on the product distribution, supports a reaction mechanism involving an acyl-enzyme intermediate whose formation is rate-determining under conditions of substrate saturation. Consideration of the variation of these kinetic parameters with the structure of the depsipeptides and comparison with the analogous parameters for bicyclic beta-lactam substrates suggest that a variety of substrate binding modes exist on this enzyme. The class A enzymes, B. cereus beta-lactamase I and the TEM-2 beta-lactamase, catalyze depsipeptide and benzylpenicillin hydrolyses but not methanolysis. The acyl-enzyme derived from both types of substrate is thus shielded from external nucleophiles; the shielding is therefore not an effect, direct or indirect, of the thiazolidinyl group in the penicilloyl-enzyme. The class A beta-lactamase of the PC1 plasmid of S. aureus is distinctly different from the above two representatives of that class, in that it does catalyze methanolysis of depsipeptides (but not of benzylpenicillin). The methanolysis kinetics suggest that deacylation is rate-determining at saturation, a conclusion supported by the demonstration of an intermediate during the hydrolysis of m-[[(phenylacetyl)glycyl]oxy]benzoate, subsequent to leaving-group departure. The beta-lactamases have thus been shown to catalyze the hydrolysis of specific depsipeptides with comparable facility to that demonstrated by D-alanyl-D-alanine carboxypeptidase/transpeptidases. The former enzymes, however, differ in being unable to cleave the analogous peptides.     

An inducible β-lactamase in a strain ofEscherichia coli

The production of beta-lactamase (penicillin/cephalosporin beta-lactam amidohydrolase, E.C.3.5.2.6) was found to be inducible in a clinically isolated strain of Escherichia coli. This is the first report of an inducible beta-lactamase in E. coli. The optimal concentration of inducer was 400 mug/ml of ml of benzylpenicillin, or 800 mug/ml of 6-aminopenicillanic acid. About fiftyfold induction was achieved. Maximum induction took ninety minutes from the time of adding the inducer. Induction was abolished by the presence of chloramphenicol(10 mug/ml). The enzyme has a molecular wieght of 23,000, and is inhibited by rho-chloromercuribenzoate and by iodine. It is active against a wide range of substrates, including cephaloridine and cloxacillin.     

On the importance of a methyl group in beta-lactamase evolution: free energy profiles and molecular modeling.

beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.