Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

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.     

pKa calculations for class A beta-lactamases: methodological and mechanistic implications.

Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.     

Crystal structure of extended-spectrum beta-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion

The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 A resolution. The result reveals that the Lys73 side chain can adopt two alternative conformations. The predominant conformation of Lys73 is different from that observed in the E166A mutant, indicating that removal of the Glu166 side chain changes the conformation of the Lys73 side chain and thus the interaction between Lys73 and Glu166. The Lys73 side chain would play an important role in proton relay, switching its conformation from one to the other depending on the circumstances. The electron density map also implies possible rotation of Ser237. Comparison of the Toho-1 structure with the structure of other class A beta-lactamases shows that the hydroxyl group of Ser237 is likely to rotate through interaction with the carboxyl group of the substrate. Another peculiarity is the existence of three sulfate ions positioned in or near the substrate-binding cavity. One of these sulfate ions is tightly bound to the active center, while the other two are held by a region of positive charge formed by two arginine residues, Arg274 and Arg276. This positively charged region is speculated to represent a pseudo-binding site of the beta-lactam antibiotics, presumably catching the methoxyimino group of the third-generation cephems prior to proper binding in the substrate-binding cleft for hydrolysis. This high-resolution structure, together with detailed kinetic analysis of Toho-1, provides a new hypothesis for the catalytic mechanism and substrate specificity of Toho-1.     

Neutron Diffraction Studies of a Class A β-Lactamase Toho-1 E166A/R274N/R276N Triple Mutant

β-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the β-lactam antibiotics is the production of β-lactamase enzymes that inactivate β-lactams by rapidly hydrolyzing the amide group of the β-lactam ring. Specifically, the class A extended-spectrum β-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a β-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.     

Comparison of beta-lactamases of classes A and D: 1.5-A crystallographic structure of the class D OXA-1 oxacillinase

The crystallographic structure of the Escherichia coli OXA-1 beta-lactamase has been established at 1.5-A resolution and refined to R = 0.18. The 28.2-kD oxacillinase is a class D serine beta-lactamase that is especially active against the penicillin-type beta-lactams oxacillin and cloxacillin. In contrast to the structures of OXA-2, OXA-10, and OXA-13 belonging to other subclasses, the OXA-1 molecule is monomeric rather than dimeric and represents the subclass characterized by an enlarged Omega loop near the beta-lactam binding site. The 6-residue hydrophilic insertion in this loop cannot interact directly with substrates and, instead, projects into solvent. In this structure at pH 7.5, carboxylation of the conserved Lys 70 in the catalytic site is observed. One oxygen atom of the carboxylate group is hydrogen bonded to Ser 120 and Trp 160. The other oxygen atom is more exposed and hydrogen bonded to the Ogamma of the reactive Ser 67. In the overlay of the class D and class A binding sites, the carboxylate group is displaced ca. 2.6 A from the carboxylate group of Glu 166 of class A enzymes. However, each group is equidistant from the site of the water molecule expected to function in hydrolysis, and which could be activated by the carboxylate group of Lys 70. In this ligand-free OXA-1 structure, no water molecule is seen in this site, so the water molecule must enter after formation of the acyl-Ser 67 intermediate.     

Structure of the SHV-1 beta-lactamase

The X-ray crystallographic structure of the SHV-1 beta-lactamase has been established. The enzyme crystallizes from poly(ethylene glycol) at pH 7 in space group P212121 with cell dimensions a = 49.6 A, b = 55.6 A, and c = 87.0 A. The structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.18 for all data in the range 8.0-1.98 A resolution. Deviations of model bonds and angles from ideal values are 0.018 A and 1.8 degrees, respectively. Overlay of all 263 alpha-carbon atoms in the SHV-1 and TEM-1 beta-lactamases results in an rms deviation of 1.4 A. Largest deviations occur in the H10 helix (residues 218-224) and in the loops between strands in the beta-sheet. All atoms in residues 70, 73, 130, 132, 166, and 234 in the catalytic site of SHV-1 deviate only 0.23 A (rms) from atoms in TEM-1. However, the width of the substrate binding cavity in SHV-1, as measured from the 104-105 and 130-132 loops on one side to the 235-238 beta-strand on the other side, is 0.7-1.2 A wider than in TEM-1. A structural analysis of the highly different affinity of SHV-1 and TEM-1 for the beta-lactamase inhibitory protein BLIP focuses on interactions involving Asp/Glu104.     

Beta-lactamase of Bacillus licheniformis 749/C. Refinement at 2 A resolution and analysis of hydration

The crystallographic and molecular structure of the class A beta-lactamase (penicillinase) of Bacillus licheniformis 749/C has been refined with X-ray diffraction data to 2 A resolution. For the 27,330 data with F greater than or equal to 3 sigma(F), the R factor is 0.15; for all 30,090 data, R is 0.16. The estimated co-ordinate error is 0.15 A. In the final model, the deviation of covalent bonds and angles from ideality is 0.012 A and 2.2 degrees, respectively. The model includes two molecules of 29,500 daltons each in the asymmetric unit of space group P2(1), 484 water molecules and two tetrahedral buffer anions. Overlay of the two protein molecules results in a root-mean-square difference of 0.17 A and 0.41 A for alpha-carbon atoms and for all atoms, respectively. Twenty-six water molecules fall within 0.25 A of matching water molecules associated with the second protein molecule. The reactive Ser70 is on a turn of 3(10) helix at the N terminus of a longer alpha-helix (72-83). The penicillin-binding site near this helix contains at least seven water molecules. Upon penicillin entry, a water molecule in the oxyanion hole, hydrogen-bonded between the N terminus of helix (80-83) and beta-strand (230-238), would be displaced by the oxygen atom of the beta-lactam carbonyl group. An unexpelled molecule of water is proposed to be the catalytic water required for penicillin hydrolysis. The water is hydrogen-bonded to Glu166, a conserved residue in all beta-lactamases, and it lies 3 A from the alpha-face of a previously modeled penicillin. The position of the water-Glu166 pair is stabilized in the active site by a cis peptide bond at Pro167.     

Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.     

Lysine-73 is involved in the acylation and deacylation of beta-lactamase

Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.     

Clavulanic acid inactivation of SHV-1 and the inhibitor-resistant S130G SHV-1 beta-lactamase. Insights into the mechanism of inhibition

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser --> Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1beta-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant beta-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 beta-lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130Gbeta-lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gbeta-lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130Gbeta-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G beta-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant beta-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit beta-lactamases.     

Refined crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.0 A resolution

The crystal structure of a class A beta-lactamase from Staphylococcus aureus PC1 has been refined at 2.0 A resolution. The resulting crystallographic R-factor (R = sigma h parallel Fo[-]Fc parallel/sigma h[Fo], where [Fo] and [Fc] are the observed and calculated structure factor amplitudes, respectively), is 0.163 for the 17,547 reflections with I greater than or equal to 2 sigma (I) within the 8.0 A to 2.0 A resolution range. The molecule consists of two closely associated domains. One domain is formed by a five-stranded antiparallel beta-sheet with three helices packing against a face of the sheet. The second domain is formed mostly by helices that pack against the second face of the sheet. The active site is located in the interface between the two domains, and many of the residues that form it are conserved in all known sequences of class A beta-lactamases. Similar to the serine proteases, an oxyanion hole is implicated in catalysis. It is formed by two main-chain nitrogen atoms, that of the catalytic seryl residue, Ser70, and that of Gln237 on an edge beta-strand of the major beta-sheet. Ser70 is interacting with another conserved seryl residue, Ser130, located between the two ammonium groups of the functionally important lysine residues, Lys73 and Lys234. Such intricate interactions point to a possible catalytic role for this second seryl residue. Another key catalytic residue is Glu166. There are several unusual structural features associated with the active site. (1) A cis peptide bond has been identified between the catalytic Glu166 and Ile167. (2) Ala69 and Leu220 have strained phi, psi dihedral angles making close contacts that restrict the conformation of the active site beta-strand involved in the formation of the oxyanion hole. (3) A buried aspartate residue, the conserved Asp233, is located next to the active site Lys234. It is interacting with another buried aspartyl residue, Asp246. An internal solvent molecule is also involved, but the rest of its interactions with the protein indicate it is not a cation. (4) Another conserved aspartyl residue that is desolvated is Asp131, adjacent to Ser130. Its charge is stabilized by interactions with four main-chain nitrogen atoms. (5) An internal cavity underneath the active site depression is filled with six solvent molecules. This, and an adjacent cavity occupied by three solvent molecules partially separate the omega-loop associated with the active site from the rest of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

pKa calculations for class A beta-lactamases: influence of substrate binding

Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.     

Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases.

The class A beta-lactamases and the transpeptidase domain of the penicillin-binding proteins (PBPs) share the same topology and conserved active-site residues. They both react with beta-lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the beta-lactams. In contrast, the deacylation of the beta-lactamases is extremely fast, resulting in a high turnover of beta-lactam hydrolysis, which confers resistance to these antibiotics. In TEM-1 beta-lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH-dependent manner, for the acidic residues. The aspartic acid-containing variant had a 110-fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.     

Structure of an extended-spectrum class A beta-lactamase from Proteus vulgaris K1

The structure of a chromosomal extended-spectrum beta-lactamase (ESBL) having the ability to hydrolyze cephalosporins including cefuroxime and ceftazidime has been determined by X-ray crystallography to 1.75 A resolution. The species-specific class A beta-lactamase from Proteus vulgaris K1 was crystallized at pH 6.25 and its structure solved by molecular replacement. Refinement of the model resulted in crystallographic R and R(free) of 16.9 % and 19.3 %, respectively. The folding of the K1 enzyme is broadly similar to that of non-ESBL TEM-type beta-lactamases (2 A rmsd for C(alpha)) and differs by only 0.35 A for all atoms of six conserved residues in the catalytic site. Other residues promoting extended-spectrum activity in K1 include the side-chains of atypical residues Ser237 and Lys276. These side-chains are linked by two water molecules, one of which lies in the position normally filled by the guanidinium group of Arg244, present in most non-ESBL enzymes but absent from K1. The ammonium group of Lys276, ca 3.5 A from the virtual Arg244 guanidinium position, may interact with polar R2 substitutents on the dihydrothiazene ring of cephalosporins.     

Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp:Asn170Gln with benzylpenicillin and cephaloridine

The serine-beta-lactamases hydrolyze beta-lactam antibiotics in a reaction that proceeds via an acyl-enzyme intermediate. The double mutation, E166D:N170Q, of the class A enzyme from Staphylococcus aureus results in a protein incapable of deacylation. The crystal structure of this beta-lactamase, determined at 2.3 A resolution, shows that except for the mutation sites, the structure is very similar to that of the native protein. The crystal structures of two acyl-enzyme adducts, one with benzylpenicillin and the other with cephaloridine, have been determined at 1.76 and 1.86 A resolution, respectively. Both acyl-enzymes show similar key features, with the carbonyl carbon atom of the cleaved beta-lactam bond covalently bound to the side chain of the active site Ser70, and the carbonyl oxygen atom in an oxyanion hole. The thiadolizine ring of the cleaved penicillin is located in a slightly different position than the dihydrothiazine ring of cephaloridine. Consequently, the carboxylate moieties attached to the rings form different sets of interactions. The carboxylate group of benzylpenicillin interacts with the side chain of Gln237. The carboxylate group of cephaloridine is located between Arg244 and Lys234 side chains and also interacts with Ser235 hydroxyl group. The interactions of the cephaloridine resemble those seen in the structure of the acyl-enzyme of beta-lactamase from Escherichia coli with benzylpenicillin. The side chains attached to the cleaved beta-lactam rings of benzylpenicillin and cephaloridine are located in a similar position, which is different than the position observed in the E. coli benzylpenicillin acyl-enzyme complex. The three modes of binding do not show a trend that explains the preference for benzylpenicillin over cephaloridine in the class A beta-lactamases. Rather, the conformational variation arises because cleavage of the beta-lactam bond provides additional flexibility not available when the fused rings are intact. The structural information suggests that specificity is determined prior to the cleavage of the beta-lactam ring, when the rigid fused rings of benzylpenicillin and cephaloridine each form different interactions with the active site.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Probing active site chemistry in SHV beta-lactamase variants at Ambler position 244. Understanding unique properties of inhibitor resistance

Inhibitor-resistant class A beta-lactamases are an emerging threat to the use of beta-lactam/beta-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A beta-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A beta-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A beta-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E beta-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k(inact) for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV beta-lactamase.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.