Abstract
TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear. The N276D mutant was constructed, purified, and kinetically characterized. It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme. Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex. The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.
Highlights
TEM-35 (inhibitor resistant TEM (IRT)·4) and TEM-36 clavulanic acid-resistant p-Iactamases have evolved from TEM-l p-Iactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276
The antibiotic disc assays of the N276D mutant show that this variant is as active as the wild type {3-lactamase against penicillins and first and second generation cephalosporins, because growth is not inhibited around the antibiotic discs
Zhou et al (1994) proposed that this substitution was sufficient to explain clavulanic acid resistance of the TEM-35 enzyme and that the N276D substitution had no contribution to this phenomenon
Summary
TEM-35 (inhibitor resistant TEM (IRT)·4) and TEM-36 (lRT-7) clavulanic acid-resistant p-Iactamases have evolved from TEM-l p-Iactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The antibiograms of the exponential cultures of these transformed strains (Table III) show that all the variant proteins are less active than the wild type TEM-1 J3-lactamase.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
