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Protein Engineering, Vol. 12, No. 9, 761-769, September 1999
© 1999 Oxford University Press

Susceptibility of ß-lactamase to core amino acid substitutions

Joseph F. Petrosino1, Matthew Baker2 and Timothy Palzkill1,3,4

1 Department of Microbiology and Immunology, 2 Structural and Computational Biology and Molecular Biophysics Program and 3 Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA

To determine which amino acids in TEM-1 ß-lactamase are important for its structure and function, random libraries were previously constructed which systematically randomized the 263 codons of the mature enzyme. A comprehensive screening of these libraries identified several TEM-1 ß-lactamase core positions, including F66 and L76, which are strictly required for wild-type levels of hydrolytic activity. An examination of positions 66 and 76 in the class A ß-lactamase gene family shows that a phenylalanine at position 66 is strongly conserved while position 76 varies considerably among other ß-lactamases. It is possible that position 76 varies in the gene family because ß-lactamase mutants with non-conservative substitutions at position 76 retain partial function. In contrast, position 66 may remain unchanged in the gene family because non-conservative substitutions at this location are detrimental for enzyme structure and function. By determining the ß-lactam resistance levels of the 38 possible mutants at positions 66 and 76 in the TEM-1 enzyme, it was confirmed that position 76 is indeed more tolerant of non-conservative substitutions. An analysis of the Protein Data Bank files for three class A ß-lactamases indicates that volume constraints at position 66 are at least partly responsible for the low tolerance of substitutions at this position.

Keywords: ß-lactamase/compensating mutations/protein core/protein evolution/thermal value

4 To whom correspondence should be addressed


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