Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β‐lactamase

Palzkill, Timothy; Le, Q Q; Venkatachalam, K.V.; LaRocco, Mark; Ocera, H.

doi:10.1111/j.1365-2958.1994.tb01011.x

Cited by 90 publications

(88 citation statements)

References 38 publications

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“…Many other TEM mutations have been constructed that are able to increase resistance to extended-spectrum cephalosporins (21)(22)(23). It remains a puzzling fact that these mutations have not yet been recovered either in clinical isolates or in stepwise in vitro selection tests.…”

Section: Discussionmentioning

confidence: 99%

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases

Blázquez

Morosini

Negri

et al. 1995

Antimicrob Agents Chemother

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By directed mutagenesis, we constructed a set of seven TEM-1 derivatives containing single replacements in each one of the amino acids substituted in naturally occurring extended-spectrum TEM ␤-lactamases. The exact contribution of each mutation to the resistance phenotype was determined. In addition, mutant enzyme production and stabilities were studied. Five of seven mutations determined to some extent variations in cephalosporin and/or monobactam activity. Dramatic changes in the hydrolysis of ceftazidime and aztreonam occurred when a serine was at position 164. Changes at positions 104, 238, and 240 showed more leaky variation in activity towards cephalosporins and aztreonam. Replacements at positions 237 and 265 caused no variation in susceptibility to cephalosporins. Interestingly, the change from Gln to Lys at position 39 found in TEM-2, classically considered a neutral change, slightly but consistently increased the MIC of ceftazidime and aztreonam. The in vitro construction of mutations appearing in naturally occurring TEM-␤-lactamases, studied in the same genetic context, may help to understand the evolution of extended-spectrum ␤-lactamases.The production of TEM-type ␤-lactamases is the most prevalent mode of resistance to ␤-lactam antibiotics. TEM-1 ␤-lactamase is considered a broad-spectrum enzyme because it hydrolyzes both penicillins and cephalosporins (4). TEM-1, however, cannot efficiently inactivate new extended-spectrum cephalosporins, such as cefotaxime and ceftazidime, and monobactams, such as aztreonam. Molecular variants of TEM-1, termed extended-spectrum ␤-lactamases, emerged and disseminated probably as a consequence of the introduction of these extended-spectrum ␤-lactam antibiotics.After years of intensive study of molecular variations involved in substrate profile alterations of TEM enzymes, it is now known that clinically resistant variants are the result of amino acid substitutions in one of several well-defined positions or of combinations of two to five of these substitutions (for a review, see reference 11). Figure 1 shows the modified positions in these resistant variants and the amino acids by which they are substituted in naturally occurring TEM-type ␤-lactamases. For each position, the replacement is with a particular amino acid, the only exception being Arg-164, which can be replaced by either Ser or His. Several authors constructed TEM-1 derivatives containing some of the abovedescribed single mutations by either oligonucleotide-directed mutagenesis (10, 30) or subcloning of fragments containing those mutations (28). Biochemical and microbiological studies have been performed with some of these mutants. Nevertheless, none of these publications includes a comparative study of the variations in ␤-lactamase phenotype and stability resulting from all seven single mutations corresponding to the seven positions modified in naturally occurring ␤-lactamases. In addition, the microbiological activities were, in each one of these studies, measured in a different genetic context (prom...

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Section: Discussionmentioning

confidence: 99%

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases

Blázquez

Morosini

Negri

et al. 1995

Antimicrob Agents Chemother

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“…The ⍀-loop motif, which includes both residues 166 (critical for deacylation) and 170, assumes different conformations in various class A enzymes (Fig. 2) and, at least in the case of a subset of the TEM mutants, it has been argued to be a mobile element (27)(28)(29). Both TEM-1 (Fig.…”

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confidence: 99%

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

Frase

Shi

Testero

et al. 2009

Journal of Biological Chemistry

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A major mechanism of bacterial resistance to ␤-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is the production of ␤-lactamases. A handful of class A ␤-lactamases have been discovered that have acquired the ability to turn over carbapenem antibiotics. This is a disconcerting development, as carbapenems are often considered last resort antibiotics in the treatment of difficult infections. The GES family of ␤-lactamases constitutes a group of extended spectrum resistance enzymes that hydrolyze penicillins and cephalosporins avidly. A single amino acid substitution at position 170 has expanded the breadth of activity to include carbapenems. The basis for this expansion of activity is investigated in this first report of detailed steady-state and pre-steady-state kinetics of carbapenem hydrolysis, performed with a class A carbapenemase. Monitoring the turnover of imipenem (a carbapenem) by GES-1 (Gly-170) revealed the acylation step as rate-limiting. GES-2 (Asn-170) has an enhanced rate of acylation, compared with GES-1, and no longer has a single rate-determining step. Both the acylation and deacylation steps are of equal magnitude. GES-5 (Ser-170) exhibits an enhancement of the rate constant for acylation by a remarkable 5000-fold, whereby the enzyme acylation event is no longer rate-limiting. This carbapenemase exhibits k cat /K m of 3 ؋ 10 5 M ؊1 s ؊1 , which is sufficient for manifestation of resistance against imipenem.

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“…The strategy in understanding how amino acid substitutions affect substrate specificity has been to first use random replacement mutagenesis to randomize three or four contiguous codons to form random libraries containing all or nearly all possible amino acid combinations for the region being randomized (19)(20)(21)(22). Then, functional mutants able to catalyze the hydrolysis of a poor substrate, ceftazidime (an extended-spectrum cephalosporin), and an excellent substrate, ampicillin (a penicillin), are isolated and sequenced.…”

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confidence: 99%

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

Petrosino

Palzkill

1996

J Bacteriol

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␤-Lactamase is a bacterial protein that provides resistance against ␤-lactam antibiotics. TEM-1 ␤-lactamase is the most prevalent plasmid-mediated ␤-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant ␤-lactamases have evolved with activity toward a variety of ␤-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant ␤-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 ␤-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 ␤-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 site 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.

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Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β‐lactamase

Cited by 90 publications

References 38 publications

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases

Single amino acid replacements at positions altered in naturally occurring extended-spectrum TEM beta-lactamases

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

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

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