An engineered Staphylococcus aureus PC1 beta-lactamase that hydrolyses third-generation cephalosporins.
The beta-lactamase from Staphylococcus aureus PC1 has been cloned into an Escherichia coli vector for site-directed mutagenesis and high-level protein expression. A mutant enzyme has been produced in which Ala238 is replaced by a serine, and Ile239 is deleted (A238S:I239del). The engineered enzyme hydrolyses third-generation cephalosporins substantially more rapidly than the parental enzyme does, while hydrolysis of benzylpenicillin is slower with the mutant than with the wild-type and native enzymes. The mutant beta-lactamase has been crystallized and the structure determined and refined at 2.8 A resolution. The disposition of the beta-strand which forms the side of the active site is altered in comparison with the native S. aureus beta-lactamase structure, widening the active site cleft and providing space to accommodate the bulky side-chains of the third-generation cephalosporins.
Zawadzke
L E
LE
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Smith
T J
TJ
Herzberg
O
O
D001483:Base Sequence; D001665:Binding Sites; D002439:Cefotaxime / Q000494:pharmacology; D002442:Ceftazidime / Q000378:metabolism; D002509:Cephaloridine / Q000378:metabolism; D002511:Cephalosporins / Q000378:metabolism*; D003001:Cloning, Molecular; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D015202:Protein Engineering; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
D002511:Cephalosporins; D002442:Ceftazidime; C054232:beta-lactamase PC1; D001618:beta-Lactamases; D002509:Cephaloridine; D002439:Cefotaxime