Effect of an Amino Acid Insertion into the Omega Loop Region of a Class C β-Lactamase on Its Substrate Specificity

The class C ␤-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum ␤-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 ␤-lactamase conferring a higher MIC of cefepime (MIC, 8 g/ml, compared with 0.5 g/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other ␤-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 ␤-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat /K m ) of the Leu-293-Pro mutant ␤-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 ␤-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant ␤-lactamase.

Section: Resultssupporting

confidence: 86%

Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

Vakulenko¹,

Golemi

Geryk³

et al. 2002

“…In another study, the AmpC enzyme in an oxyiminocephalosporin-resistant clinical isolate of S. marcescens was found to contain a Glu-to-Lys change in residue 213, closer to the center of the omega loop (22). Finally, extension of the omega loop in the middle of both natural and laboratory-generated E. cloacae mutant enzymes was shown to result in an increase in k cat values to oxyiminocephalosporins, including ceftazidime (33,34). In this case, the crystal structure of the mutant enzyme indeed showed the increased opening of the entrance of the substrate-binding pocket (4).…”

Section: Properties Of the Wild-type Enzyme And Their Consequencesmentioning

confidence: 89%

Mutation in Serratia marcescens AmpC β-Lactamase Producing High-Level Resistance to Ceftazidime and Cefpirome

Raimondi

Sisto

Nikaido

2001

Starting from a clinical isolate of Serratia marcescens that produced a chromosomally encoded AmpC ␤-lactamase inducibly, we isolated by stepwise selection two laboratory mutants that showed high levels of resistance to some cephalosporins. The 98R mutant apparently overproduced the unaltered ␤-lactamase constitutively, but the 520R mutant produced an altered enzyme, also constitutively. Ceftazidime and cefpirome MICs for the 520R mutant were much higher (512 and 64 g/ml, respectively) than those for the 98R mutant (16 and 16 g/ml, respectively). Yet the MICs of cephaloridine and piperacillin for the 520R mutant were four-to eightfold lower than those for the 98R mutant. Cloning and sequencing of the ampC alleles showed that in the 520R mutant enzyme, the Thr64 residue, about two turns away from the active-site serine, was mutated to isoleucine. This resulted in a >1,000-fold increase in the catalytic efficiency (k cat /K m ) of the mutated AmpC enzyme toward ceftazidime, whereas there was a >10-fold decrease in the efficiency of the mutant enzyme toward cefazolin and cephaloridine. The outer membrane permeability of the 520R strain to cephalosporins was also less than in the 98R strain, and the alteration of the kinetic properties of the AmpC enzyme together with this difference in permeability explained quantitatively the resistance levels of both mutant strains to most agents studied.For many gram-negative bacteria, including Enterobacteriaceae and Pseudomonas spp., the production of the chromosomally encoded, class C ␤-lactamase, or the AmpC enzyme, represents the intrinsic mechanism of resistance to ␤-lactam antibiotics. AmpC expression is under the control of a regulatory gene system. Spontaneous mutations affecting the regulatory genes, most frequently ampD (17), cause constitutive overproduction of the enzyme and an increased resistance to agents, such as oxyiminocephalosporins (cefotaxime, cefuroxime, ceftriaxone, and ceftazidime) (38). Most of these compounds are hydrolyzed efficiently because of their high affinity for the AmpC enzyme, which compensates for their low deacylation rates (8,12,49); yet against the wild-type strains of Enterobacteriaceae, these compounds are quite effective because they are poor inducers of AmpC (18,39). Additionally, mutations in ␤-lactamase structural genes may also confer a modified spectrum of drug resistance to the producing organism. Spontaneous mutations occurring in plasmid-encoded ␤-lactamases, resulting in the production of expanded-spectrum ␤-lactamases, are of great concern since they can be spread efficiently through the plasmid transfer process (25).Until now, mutations in structural genes of chromosomal ␤-lactamases have been reported in only a few cases. Thus, clinical strains of Enterobacter cloacae (33) and Serratia marcescens (22) showing increased resistance to oxyiminocephalosporins, especially ceftazidime, were found to produce chromosomal AmpC enzymes with alterations in the "omega loop," located at the entrance of the substrate-binding sites. It is...

“…Extension of the ⍀-loop for AmpC ␤-lactamase from Enterobacter cloacae has been shown to broaden its hydrolysis spectrum (15,16). In that case, the mutant enzyme exhibited an increased opening of the entrance of the substrate-binding pocket (6).…”

Section: Mic (G/ml)mentioning

confidence: 99%

Extended-Spectrum Cephalosporinase in Acinetobacter baumannii

Rodríguez-Martínez

Nordmann

Ronco

et al. 2010

An AmpC-type ␤-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C ␤-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the ⍀-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.