Analysis of the context dependent sequence requirements of active site residues in the metallo-beta-lactamase IMP-1

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.     

Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.     

Impact of remote mutations on metallo-beta-lactamase substrate specificity: implications for the evolution of antibiotic resistance

Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.     

Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase.

TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-1 gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 beta-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin.     

Amino acid sequence determinants of extended spectrum cephalosporin hydrolysis by the class C P99 beta-lactamase

Class C beta-lactamases are commonly encoded on the chromosome of Gram-negative bacterial species. Mutations leading to increased expression of these enzymes are a common cause of resistance to many cephalosporins including extended spectrum cephalosporins. Recent reports of plasmid- and integrin-encoded class C beta-lactamases are a cause for concern because these enzymes are likely to spread horizontally to susceptible strains. Because of their increasing clinical significance, it is critical to identify the determinants of catalysis and substrate specificity of these enzymes. For this purpose, the codons of a set of 21 amino acid residues that encompass the active site region of the P99 beta-lactamase were individually randomized to create libraries containing all possible amino acid substitutions. The amino acid sequence requirements for the hydrolysis of ceftazidime, an extended spectrum cephalosporin commonly used to treat serious infections, were determined by selecting resistant mutants from each of the 21 libraries. DNA sequencing identified the residue positions that are critical for ceftazidime hydrolysis. In addition, it was found that certain amino acid substitutions in the omega-loop region of the P99 enzyme result in increased ceftazidime hydrolysis suggesting the loop is an important determinant of substrate specificity.     

Susceptibility of beta-lactamase to core amino acid substitutions.

To determine which amino acids in TEM-1 beta-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 beta-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A beta-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other beta-lactamases. It is possible that position 76 varies in the gene family because beta-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the beta-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A beta-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.     

Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.     

Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae

We have determined the nucleotide sequence of the plasmid genes blaT-4 and blaT-5 which encode the broad-substrate-range beta-lactamases TEM-4 and TEM-5, respectively. The TEM-4 enzyme, which confers high-level resistance to cefotaxime (Ctx) and ceftazidime (Caz), differed from the TEM-1 penicillinase by four amino acid substitutions. Two of the mutations are identical to those responsible for the wide substrate range of the TEM-3 beta-lactamase which hydrolyses Ctx and Caz. The amino acid sequence of TEM-5, which confers higher levels of resistance to Caz than to other recently developed cephalosporins, differed from that of TEM-1 by three mutations distinct from those of TEM-4. Analysis of the location of the mutations in the primary and tertiary structures of class A beta-lactamases suggests that interactions between the substituted residues and beta-lactam antibiotics non-hydrolysable by TEM-1 and TEM-2 allow TEM-4 and TEM-5 to hydrolyse efficiently novel broad-spectrum cephalosporins such as Ctx and Caz.     

Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat Ends

A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ' process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val(72), Ser(153), Lys(160)-Ile(161), Gly(163)-Val(165), and His(171)-Leu(172). Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser(153) and Val(72) in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance.     

A highly active adipyl-cephalosporin acylase obtained via rational randomization

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY-77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl-7-aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine-deficient Escherichia coli strain and subjected to growth selection with adipyl-leucine or amino-adipyl-leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl-7-ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl-7-ADCA 36-fold. The activity of this mutant towards adipyl-7-ADCA is 50% of the activity of the wild-type enzyme towards the preferred substrate glutaryl-7-aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.     

Understanding the determinants of substrate specificity in IMP family metallo-β-lactamases: The importance of residue 262

In Gram-negative bacteria, resistance to β-lactam antibacterials is largely due to β-lactamases and is a growing public health threat. One of the most concerning β-lactamases to evolve in bacteria are the Class B enzymes, the metallo-β-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel β-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of β-lactam antibacterials. Catalytic efficiencies (k_cat/K_M) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in β-lactam turnover and support this approach to predict activities of certain novel MBL variants.     

Mutagenic mapping of helical structures in the transmembrane segments of the yeast alpha-factor receptor

The alpha-mating pheromone receptor encoded by the yeast STE2 gene is a G protein coupled receptor that initiates signaling via a MAP kinase pathway that prepares haploid cells for mating. To establish the range of allowed amino acid substitutions within transmembrane segments of this receptor, we conducted extensive random mutagenesis of receptors followed by screening for receptor function. A total of 157 amino acid positions in seven different mutagenic libraries corresponding to the seven predicted transmembrane segments were analyzed, yielding 390 alleles that retain at least 60 % of normal signaling function. These alleles contained a total of 576 unique amino acid substitutions, including 61 % of all the possible amino acid changes that can arise from single base substitutions. The receptor exhibits a surprising tolerance for amino acid substitutions. Every amino acid in the mutagenized regions of the transmembrane regions could be substituted by at least one other residue. Polar amino acids were tolerated in functional receptors at 115 different positions (73 % of the total). Hydrophobic amino acids were tolerated in functional receptors at all mutagenized positions. Substitutions introducing proline residues were recovered at 53 % of all positions where they could be brought about by single base changes. Residues with charged side-chains could also be tolerated at 53 % of all positions where they were accessible through single base changes. The spectrum of allowed amino acid substitutions was characterized in terms of the hydrophobicity, radius of gyration, and charge of the allowed substitutions and mapped onto alpha-helical structures. By comparing the patterns of allowed substitutions with the recently determined structure of rhodopsin, structural features indicative of helix-helix interactions can be discerned in spite of the extreme sequence divergence between these two proteins.     

Expanded molecular diversity generation during directed evolution by trinucleotide exchange (TriNEx)

Trinucleotide exchange (TriNEx) is a method for generating novel molecular diversity during directed evolution by random substitution of one contiguous trinucleotide sequence for another. Single trinucleotide sequences were deleted at random positions in a target gene using the engineered transposon MuDel that were subsequently replaced with a randomized trinucleotide sequence donated by the DNA cassette termed SubSeq(NNN). The bla gene encoding TEM-1 beta-lactamase was used as a model to demonstrate the effectiveness of TriNEx. Sequence analysis revealed that the mutations were distributed throughout bla, with variants containing single, double and triple nucleotide changes. Many of the resulting amino acid substitutions had significant effects on the in vivo activity of TEM-1, including up to a 64-fold increased activity toward ceftazidime and up to an 8-fold increased resistance to the inhibitor clavulanate. Many of the observed amino acid substitutions were only accessible by exchanging at least two nucleotides per codon, including charge-switch (R164D) and aromatic substitution (W165Y) mutations. TriNEx can therefore generate a diverse range of protein variants with altered properties by combining the power of site-directed saturation mutagenesis with the capacity of whole-gene mutagenesis to randomly introduce mutations throughout a gene.     

An analysis of why highly similar enzymes evolve differently

The TEM-1 and SHV-1 beta-lactamases are important contributors to resistance to beta-lactam antibiotics in gram-negative bacteria. These enzymes share 68% amino acid sequence identity and their atomic structures are nearly superimposable. Extended-spectrum cephalosporins were introduced to avoid the action of these beta-lactamases. The widespread use of antibiotics has led to the evolution of variant TEM and SHV enzymes that can hydrolyze extended-spectrum antibiotics. Despite being highly similar in structure, the TEM and SHV enzymes have evolved differently in response to the selective pressure of antibiotic therapy. Examples of this are at residues Arg164 and Asp179. Among TEM variants, substitutions are found only at position 164, while among SHV variants, substitutions are found only at position 179. To explain this observation, the effects of substitutions at position 164 in both TEM-1 and SHV-1 on antibiotic resistance and on enzyme catalytic efficiency were examined. Competition experiments were performed between mutants to understand why certain substitutions preferentially evolve in response to the selective pressure of antibiotic therapy. The data presented here indicate that substitutions at position Asp179 in SHV-1 and Arg164 in TEM-1 are more beneficial to bacteria because they provide increased fitness relative to either wild type or other mutants.     

Modeling domino effects in enzymes: molecular basis of the substrate specificity of the bacterial metallo-beta-lactamases IMP-1 and IMP-6

Metallo-beta-lactamases can hydrolyze a broad spectrum of beta-lactam antibiotics and thus confer resistance to bacteria. For the Pseudomonas aeruginosa enzyme IMP-1, several variants have been reported. IMP-6 and IMP-1 differ by a single residue (glycine and serine at position 196, respectively), but have significantly different substrate spectra; while the catalytic efficiency toward the two cephalosporins cephalothin and cefotaxime is similar for both variants, IMP-1 is up to 10-fold more efficient than IMP-6 toward cephaloridine and ceftazidime. Interestingly, this biochemical effect is caused by a residue remote from the active site. The substrate-specific impact of residue 196 was studied by molecular dynamics simulations using a cationic dummy atom approach for the zinc ions. Substrates were docked in an intermediate structure near the transition state to the binding site of IMP-1 and IMP-6. At a simulation temperature of 100 K, most complexes were stable during 1 ns of simulation time. However, at higher temperatures, some complexes became unstable and the substrate changed to a nonactive conformation. To model stability, six molecular dynamics simulations at 100 K were carried out for all enzyme-substrate complexes. Stable structures were further heated to 200 and 300 K. By counting stable structures, we derived a stability ranking score which correlated with experimentally determined catalytic efficiency. The use of a stability score as an indicator of catalytic efficiency of metalloenzymes is novel, and the study of substrates in a near-transition state intermediate structure is superior to the modeling of Michaelis complexes. The remote effect of residue 196 can be described by a domino effect: upon replacement of serine with glycine, a hole is created and a stabilizing interaction between Ser196 and Lys33 disappears, rendering the neighboring residues more flexible; this increased flexibility is then transferred to the active site.     

Evidence for frequent OspC gene transfer between Borrelia valaisiana sp. nov. and other Lyme disease spirochetes

IMP-1 metallo-beta-lactamase is a transferable carbapenem-hydrolyzing enzyme found in some clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Bacteria that express IMP-1 show significantly reduced sensitivity to carbapenems and other beta-lactam antibiotics. A series of thioester derivatives has been shown to competitively inhibit purified IMP-1. As substrates for IMP-1, the thioesters yielded thiol hydrolysis products which themselves were reversible competitive inhibitors. The thioesters also increased sensitivity to the carbapenem L-742,728 in an IMP-1-producing laboratory stain of Escherichia coli, but will need further modification to improve their activity in less permeable organisms such as Pseudomonas and Serratia. Nonetheless, the thioester IMP-1 inhibitors offer an encouraging start to overcoming metallo-beta-lactamase-mediated resistance in bacteria.     

Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae.

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.     

Identification of residues critical for catalysis in a class C beta-lactamase by combinatorial scanning mutagenesis

Despite their clinical importance, the mechanism of action of the class C beta-lactamases is poorly understood. In contrast to the class A and class D beta-lactamases, which contain a glutamate residue and a carbamylated lysine in their respective active sites that are thought to serve as general base catalysts for beta-lactam hydrolysis, the mechanism of activation of the serine and water nucleophiles in the class C enzymes is unclear. To probe for residues involved in catalysis, the class C beta-lactamase from Enterobacter cloacae P99 was studied by combinatorial scanning mutagenesis at 122 positions in and around the active site. Over 1000 P99 variants were screened for activity in a high-throughput in vivo antibiotic resistance assay and sequenced by 96-capillary electrophoresis to identify residues that are important for catalysis. P99 mutants showing reduced capability to convey antibiotic resistance were purified and characterized in vitro. The screen identified an active-site hydrogen-bonding network that is key to catalysis. A second cluster of residues was identified that likely plays a structural role in the enzyme. Otherwise, residues not directly contacting the substrate showed tolerance to substitution. The study lends support to the notion that the class C beta-lactamases do not have a single residue that acts as the catalytic general base. Rather, catalysis is affected by a hydrogen-bonding network in the active site, suggesting a possible charge relay system.     

Twelve positions in a β-lactamase that can expand its substrate spectrum with a single amino acid substitution

The continuous evolution of β-lactamases resulting in bacterial resistance to β-lactam antibiotics is a major concern in public health, and yet the underlying molecular basis or the pattern of such evolution is largely unknown. We investigated the mechanics of the substrate fspectrum expansion of the class A β-lactamase using PenA of Burkholderia thailandensis as a model. By analyzing 516 mutated enzymes that acquired the ceftazidime-hydrolyzing activity, we found twelve positions with single amino acid substitutions (altogether twenty-nine different substitutions), co-localized at the active-site pocket area. The ceftazidime MIC (minimum inhibitory concentration) levels and the relative frequency in the occurrence of substitutions did not correlate well with each other, and the latter appeared be largely influenced by the intrinsic mutational biases present in bacteria. Simulation studies suggested that all substitutions caused a congruent effect, expanding the space in a conserved structure called the omega loop, which in turn increased flexibility at the active site. A second phase of selection, in which the mutants were placed under increased antibiotic pressure, did not result in a second mutation in the coding region, but a mutation that increased gene expression arose in the promoter. This result suggests that the twelve amino acid positions and their specific substitutions in PenA may represent a comprehensive repertoire of the enzyme's adaptability to a new substrate. These mapped substitutions represent a comprehensive set of general mechanical paths to substrate spectrum expansion in class A β-lactamases that all share a functional evolutionary mechanism using common conserved residues.     

Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin

The location of amino acid substitutions that allow an enzyme to discriminate between the binding of its normal substrate and a substrate analogue may be used to identify regions of the polypeptide that fold to form the substrate binding site. We have isolated a large number of cephalexin-resistant mutants of Escherichia coli in which the resistance is due to the production of altered forms of penicillin-binding protein 3 that have reduced affinity for the antibiotic. Using three mutagens, and a variety of selection procedures, we obtained only five classes of mutants which could be distinguished by their patterns of cross-resistance to other beta-lactam antibiotics. The three classes of mutants that showed the highest levels of resistance to cephalexin were cross-resistant to several other cephalosporins but not to penicillins or to the monobactam, aztreonam. The penicillin-binding protein 3 gene from 46 independent mutants was cloned and sequenced. Each member of the five classes of cephalexin-resistant mutants had the same amino acid substitution in penicillin-binding protein 3. The mutants that showed the highest levels of resistance to cephalexin had alterations of either Thr-308 to Pro, Val-344 to Gly, or Asn-361 to Ser. The Thr-308 to Pro substitution had occurred within the beta-lactam-binding site since the adjacent residue (Ser-307) has been shown to be acylated by benzylpenicillin. The Asn-361 to Ser change occurred in a region that showed substantial similarity to regions in both penicillin-binding protein 1A and 1B and may also define a residue that is located within the beta-lactam-binding site in the three-dimensional structure of the enzyme.