Insertion mutagenesis as a tool in the modification of protein function. Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase.
The TEM-1 beta-lactamase enzyme efficiently hydrolyzes beta-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant beta-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of beta-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant beta-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Omega-loop region, which forms part of the catalytic pocket of the beta-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Omega-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Omega-loop with regards the primary structure requirements for ceftazidime hydrolysis by beta-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed.
Hayes
F
F
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. fhayes@worf.molbiol.ox.ac.uk
Hallet
B
B
Cao
Y
Y
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001618:beta-Lactamases; C034413:beta-lactamase TEM-1