Molecular evolution of a class C beta-lactamase extending its substrate specificity

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Enterobacter cloacae produces various beta-lactamases, enzymes conferring antibiotic resistance.
• Class C beta-lactamases are significant in bacterial resistance to beta-lactam antibiotics.
• The P99 beta-lactamase is a well-characterized E. cloacae enzyme.

Purpose of the Study:

• To characterize a novel class C beta-lactamase from Enterobacter cloacae GC1.
• To determine the genetic basis for its extended substrate specificity.
• To elucidate the role of specific amino acid sequences in enzyme function.

Main Methods:

• DNA sequencing of the GC1 beta-lactamase gene.
• Amino acid sequence analysis and comparison with known beta-lactamases.
• Construction and characterization of chimeric and mutant beta-lactamases using site-directed mutagenesis.

Main Results:

• Enterobacter cloacae GC1 produces a class C beta-lactamase with extended substrate specificity against oxyimino beta-lactam antibiotics.
• A unique 3-amino acid duplication (Ala-Val-Arg-Ala-Val-Arg) was identified in the GC1 enzyme, resulting from a 9-nucleotide tandem duplication.
• Chimeric and mutant enzyme analyses confirmed that the 3-amino acid insertion is solely responsible for the extended substrate specificity.

Conclusions:

• The extended substrate specificity of the GC1 beta-lactamase is attributed to a novel 3-amino acid insertion.
• This insertion, independent of amino acid characteristics, significantly enhances the enzyme's ability to hydrolyze oxyimino beta-lactam antibiotics.
• Understanding this mechanism provides insights into the evolution of antibiotic resistance in Enterobacter cloacae.