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The Role of Rigid Residues in Modulating TEM-1 β-Lactamase Function and Thermostability.

ABSTRACT: The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.

SUBMITTER: Kolbaba-Kartchner B

PROVIDER: S-EPMC7999226 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Enzyme Efficiency but Not Thermostability Drives Cefotaxime Resistance Evolution in TEM-1 β-Lactamase.

Project description:A leading intellectual challenge in evolutionary genetics is to identify the specific phenotypes that drive adaptation. Enzymes offer a particularly promising opportunity to pursue this question, because many enzymes' contributions to organismal fitness depend on a comparatively small number of experimentally accessible properties. Moreover, on first principles the demands of enzyme thermostability stand in opposition to the demands of catalytic activity. This observation, coupled with the fact that enzymes are only marginally thermostable, motivates the widely held hypothesis that mutations conferring functional improvement require compensatory mutations to restore thermostability. Here, we explicitly test this hypothesis for the first time, using four missense mutations in TEM-1 β-lactamase that jointly increase cefotaxime Minimum Inhibitory Concentration (MIC) ∼1500-fold. First, we report enzymatic efficiency (kcat/KM) and thermostability (Tm, and thence ΔG of folding) for all combinations of these mutations. Next, we fit a quantitative model that predicts MIC as a function of kcat/KM and ΔG. While kcat/KM explains ∼54% of the variance in cefotaxime MIC (∼92% after log transformation), ΔG does not improve explanatory power of the model. We also find that cefotaxime MIC rises more slowly in kcat/KM than predicted. Several explanations for these discrepancies are suggested. Finally, we demonstrate substantial sign epistasis in MIC and kcat/KM, and antagonistic pleiotropy between phenotypes, in spite of near numerical additivity in the system. Thus constraints on selectively accessible trajectories, as well as limitations in our ability to explain such constraints in terms of underlying mechanisms are observed in a comparatively "well-behaved" system.

| S-EPMC5400381 | biostudies-literature

Role of Synonymous Mutations in the Evolution of TEM β-Lactamase Genes.

Project description:Nonsynonymous mutations are well documented in TEM β-lactamases. The resulting amino acid changes often alter the conferred phenotype from broad spectrum (2b) conferred by TEM-1 to extended spectrum (2be), inhibitor resistant (2br), or both extended spectrum and inhibitor resistant (2ber). The encoding blaTEM genes also deviate in numerous synonymous mutations, which are not well understood. blaTEM-3 (2be), blaTEM-33 (2br), and blaTEM-109 (2ber) were studied in comparison to blaTEM-1blaTEM-33 was chosen for more detailed studies because it deviates from blaTEM-1 by a single nonsynonymous mutation and three additional synonymous mutations. Genes encoding the enzymes with only nonsynonymous or all (including synonymous) mutations plus all permutations between blaTEM-1 and blaTEM-33 were expressed in Escherichia coli cells. In disc diffusion assays, genes encoding TEM-3, TEM-33, and TEM-109 with all synonymous mutations resulted in higher resistance levels than genes without synonymous mutations. Disc diffusion assays with the 16 genes carrying all possible nucleotide change combinations between blaTEM-1 and blaTEM-33 indicated different susceptibilities for different variants. Nucleotide BLAST searches did not identify genes without synonymous mutations but did identify some without nonsynonymous mutations. Energies of possible secondary mRNA structures calculated with mfold are generally higher with synonymous mutations, suggesting that their role could be to destabilize the mRNA and facilitate its unfolding for efficient translation. In summary, our data indicate that transition from blaTEM-1 to other variant genes by simply acquiring the nonsynonymous mutations is not favored. Instead, synonymous mutations seem to support the transition to other variant genes with nonsynonymous mutations leading to different phenotypes.

| S-EPMC8315918 | biostudies-literature

Updated sequence information for TEM beta-lactamase genes.

Project description:The sequences of the promoter regions and of the structural genes for 13 penicillinase, extended-spectrum, and inhibitor-resistant TEM-type beta-lactamases have been determined, and an updated blaTEM gene nomenclature is proposed.

| S-EPMC89080 | biostudies-literature

Role of pleiotropy during adaptation of TEM-1 β-lactamase to two novel antibiotics.

Project description:Pleiotropy is a key feature of the genotype-phenotype map, and its form and extent have many evolutionary implications, including for the dynamics of adaptation and the evolution of specialization. Similarly, pleiotropic effects of antibiotic resistance mutations may affect the evolution of antibiotic resistance in the simultaneous or fluctuating presence of different antibiotics. Here, we study the role of pleiotropy during the in vitro adaptation of the enzyme TEM-1 β-lactamase to two novel antibiotics, cefotaxime (CTX) and ceftazidime (CAZ). We subject replicate lines for four rounds of evolution to selection with CTX and CAZ alone, and in their combined and fluctuating presence. Evolved alleles show positive correlated responses when selecting with single antibiotics. Nevertheless, pleiotropic constraints are apparent from the effects of single mutations and from selected alleles showing smaller correlated than direct responses and smaller responses after simultaneous and fluctuating selection with both than with single antibiotics. We speculate that these constraints result from structural changes in the oxyanion pocket surrounding the active site, where accommodation of CTX and the larger CAZ is balanced against their positioning with respect to the active site. Our findings suggest limited benefits from the combined or fluctuating application of these related cephalosporins for containing antibiotic resistance.

| S-EPMC4380919 | biostudies-literature

CMT-type beta-lactamase TEM-125, an emerging problem for extended-spectrum beta-lactamase detection.

Project description:The clinical strain Escherichia coli TO799 was resistant to penicillin-clavulanate combinations and ceftazidime and was not reproducibly detected as an extended-spectrum beta-lactamase (ESBL) according to the standards of the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS) and the national guidelines of the French Society for Microbiology (Comité de l'Antibiogramme de la Société Française de Microbiologie). A novel beta-lactamase, designated TEM-125, was responsible for this phenotype. TEM-125 harbors a complex association of mutations previously described in the ESBL TEM-12 and in the inhibitor-resistant beta-lactamase TEM-39. TEM-125 is the first complex mutant TEM to present hydrolytic activity against ceftazidime (kcat, 3.7 s(-1)) together with a high level of resistance to clavulanate (50% inhibitory concentration, 13.6 microM). The discovery of such an ESBL, which is difficult to detect by the usual ESBL detection methods, confirms the emergence of a complex mutant TEM subgroup and highlights the need to evaluate detection methods so as to avoid possible therapeutic failures.

| S-EPMC1489774 | biostudies-literature

TEM-121, a novel complex mutant of TEM-type beta-lactamase from Enterobacter aerogenes.

Project description:Enterobacter aerogenes clinical isolate LOR was resistant to penicillins and ceftazidime but susceptible to cefuroxime, cephalothin, cefoxitin, cefotaxime, ceftriaxone, and cefepime. PCR and cloning experiments from this strain identified a novel TEM-type beta-lactamase (TEM-121) differing by five amino acid substitutions from beta-lactamase TEM-2 (Glu104Lys, Arg164Ser, Ala237Thr, Glu240Lys, and Arg244Ser) and by only one amino acid change from the extended-spectrum beta-lactamase (ESBL) TEM-24 (Arg244Ser), with the last substitution also being identified in the inhibitor-resistant beta-lactamase IRT-2. Kinetic parameters indicated that TEM-121 hydrolyzed ceftazidime and aztreonam (like TEM-24) and was inhibited weakly by clavulanic acid and strongly by tazobactam. Thus, TEM-121 is a novel complex mutant TEM beta-lactamase (CMT-4) combining the kinetic properties of an ESBL and an inhibitor-resistant TEM enzyme.

| S-EPMC529203 | biostudies-literature

Novel TEM-type extended-spectrum beta-lactamase, TEM-134, in a Citrobacter koseri clinical isolate.

Project description:A new natural TEM derivative with extended-spectrum beta-lactamase activity, TEM-134, was identified in a ceftazidime-resistant clinical isolate of Citrobacter koseri. Compared to TEM-1, TEM-134 contains the following mutations: Q39K, E104K, R164H, and G238S. The bla(TEM-134) gene was not transferable by conjugation and, apparently, was chromosomally encoded. Expression studies with Escherichia coli revealed efficient cefotaximase and ceftazidimase activity for TEM-134.

| S-EPMC1068604 | biostudies-literature

A novel extended-spectrum TEM-type beta-lactamase, TEM-138, from Salmonella enterica serovar Infantis.

Project description:A novel natural TEM beta-lactamase with extended-spectrum activity, TEM-138, was identified in a ceftazidime-resistant clinical isolate of Salmonella enterica serovar Infantis. Compared to TEM-1, TEM-138 contains the following mutations: E104K, N175I, and G238S. The bla(TEM-138) gene was located on a 50-kb transferable plasmid. Expression studies with Escherichia coli revealed efficient ceftazidimase and cefotaximase activities for TEM-138.

| S-EPMC1563563 | biostudies-literature

Fine mapping of the sequence requirements for binding of beta-lactamase inhibitory protein (BLIP) to TEM-1 beta-lactamase using a genetic screen for BLIP function.

Project description:Beta-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases with a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for binding the TEM-1, SME-1, SHV-1, and Bla1 beta-lactamases. Twenty-three BLIP residues that contact TEM-1 beta-lactamase in the structure of the complex were mutated to alanine and assayed for inhibition (K(i)) of beta-lactamase to identify two hotspots of binding energy. These studies have been extended by the development of a genetic screen for BLIP function in Escherichia coli. The bla(TEM-1) gene encoding TEM-1 beta-lactamase was inserted into the E. coli pyrF chromosomal locus. Expression of wild-type BLIP from a plasmid in this strain resulted in a large decrease in ampicillin resistance, while introduction of the same plasmid lacking BLIP had no effect on ampicillin resistance. In addition, it was found that when the BLIP alanine-scanning mutants were tested in the strain, the level of ampicillin resistance was proportional to the K(i) of the BLIP mutant. These results indicate that BLIP function can be monitored by the level of ampicillin resistance of the genetic test strain. Each of the 23 BLIP positions examined by alanine scanning was randomized to create libraries containing all possible substitutions at each position. The genetic screen for BLIP function was used to sort the libraries for active mutants, and DNA sequence analysis of functional BLIP mutants identified the sequences required for binding TEM-1 beta-lactamase. The results indicate the BLIP surface is tolerant of substitutions in that many contact positions can be substituted with other amino acid types and retain wild-type levels of function.

| S-EPMC2728291 | biostudies-literature

Quantifying nonspecific TEM beta-lactamase (blaTEM) genes in a wastewater stream.

Project description:To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are beta-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by beta-lactamases. Of the beta-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM beta-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla(TEM) genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla(TEM) are more likely to survive the treatment process. Thus, beta-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.

| S-EPMC2612200 | biostudies-literature

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