Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130.
Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.
Helfand
Marion S
MS
Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Bethel
Christopher R
CR
Hujer
Andrea M
AM
Hujer
Kristine M
KM
Anderson
Vernon E
VE
Bonomo
Robert A
RA
D000667:Ampicillin / Q000494:pharmacology; D001667:Binding, Competitive; D019818:Clavulanic Acid / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D000078142:Tazobactam; D013816:Thermodynamics; D013997:Time Factors; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; D047090:beta-Lactams / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D019818:Clavulanic Acid; D012694:Serine; D000667:Ampicillin; D010397:Penicillanic Acid; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D000078142:Tazobactam