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.     

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.     

Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.     

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.     

Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.     

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.     

An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins

Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins. To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted. Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3. The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant. Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme. This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1. Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail. The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop. The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1.     

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.     

Molecular mechanism of ADP-ribose hydrolysis by human NUDT5 from structural and kinetic studies

Human NUDT5 (hNUDT5) is an ADP-ribose (ADPR) pyrophosphatase (ADPRase) that plays important roles in controlling the intracellular levels of ADPR and preventing non-enzymatic ADP-ribosylation of proteins by hydrolyzing ADPR to AMP and ribose 5′-phosphate. We report the crystal structure of hNUDT5 in complex with a non-hydrolyzable ADPR analogue, α,β-methyleneadenosine diphosphoribose, and three Mg^2 + ions representing the transition state of the enzyme during catalysis. Analysis of this structure and comparison with previously reported hNUDT5 structures identify key residues involved in substrate binding and catalysis. In the transition-state structure, three metal ions are bound at the active site and are coordinated by surrounding residues and water molecules. A conserved water molecule is at an ideal position for nucleophilic attack on the α-phosphate of ADPR. The side chain of Glu166 on loop L9 changes its conformation to interact with the conserved water molecule compared with that in the substrate-bound structure and appears to function as a catalytic base. Mutagenesis and kinetic studies show that Trp28 and Trp46 are important for the substrate binding; Arg51 is involved in both the substrate binding and the catalysis; and Glu112 and Glu116 of the Nudix motif, Glu166 on loop L9, and Arg111 are critical for the catalysis. The structural and biochemical data together reveal the molecular basis of the catalytic mechanism of ADPR hydrolysis by hNUDT5. Specifically, Glu166 functions as a catalytic base to deprotonate a conserved water molecule that acts as a nucleophile to attack the α-phosphate of ADPR, and three Mg^2 + ions are involved in the activation of the nucleophile and the binding of the substrate. Structural comparison of different ADPRases also suggests that most dimeric ADPRases may share a similar catalytic mechanism of ADPR hydrolysis.     

Theoretical studies of the ATP hydrolysis mechanism of myosin

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

The crystal structure of seabream antiquitin reveals the structural basis of its substrate specificity

The crystal structure of seabream antiquitin in complex with the cofactor NAD(+) was solved at 2.8A resolution. The mouth of the substrate-binding pocket is guarded by two conserved residues, Glu120 and Arg300. To test the role of these two residues, we have prepared the two mutants E120A and R300A. Our model and kinetics data suggest that antiquitin's specificity towards the substrate alpha-aminoadipic semialdehyde is contributed mainly by Glu120 which interacts with the alpha-amino group of the substrate. On the other hand, Arg300 does not have any specific interaction with the alpha-carboxylate group of the substrate, but is important in maintaining the active site conformation.     

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.     

Arginine coordination in enzymatic phosphoryl transfer: evaluation of the effect of Arg166 mutations in Escherichia coli alkaline phosphatase

Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by approximately 3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 A X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.     

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.     

Site-directed mutagenesis of residues 164, 170, 171, 179, 220, 237 and 242 in PER-1 beta-lactamase hydrolysing expanded-spectrum cephalosporins.

The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), is characterized by a substrate profile similar to that conferred by these latter enzymes. The role of residues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER-1, was assessed by site-directed mutagenesis. Replacement of Ala164 by Arg yielded an enzyme with no detectable beta-lactamase activity. Two other mutants, N179D and A164R+N179D, were also inactive. Conversely, a mutant with the A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme. Moreover, the replacement of Ala171 by Glu in the A164R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E enzyme. A specific increase in kcat for cefotaxime was observed with H170N, whereas R220L and T237A displayed a specific decrease in activity towards the same drug and a general increase in affinity towards cephalosporins. Finally, the K242E mutant displayed a kinetic behaviour very similar to that of PER-1. Based on three-dimensional models generated by homology modelling and molecular dynamics, these results suggest novel structure-activity relationships in PER-1, when compared with those previously described for the TEM-type ESBLs.     

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)     

Structural basis of phospholipase activity of Staphylococcus hyicus lipase

Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.