Site Saturation Mutagenesis of Residue 71 in β-Lactamase

Citation

Schultz, Steve Chester (1986) Site Saturation Mutagenesis of Residue 71 in β-Lactamase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/c3a0-kd54. https://resolver.caltech.edu/CaltechTHESIS:10232019-174017653

Abstract

A method was developed to "saturate" a particular residue in a protein with all possible amino acid substitutions. The method essentially involves introducing into the gene at the codon for the residue of interest a mixture of nucleotides: A, C, G, and T at the first two positions, C and G at the third position. This mixture of oligonucleotides can be readily introduced into the gene as a cassette when restriction sites are conveniently nearby. This method was used to saturate residue 71 in the pBR322 encoded β-lactamase. The first step was introduction of restriction sites into pBR322 that flank the codon for residue 71: an Ava I site at 3972 and a Sca I site at 3937. The DNA fragment between these residues was removed and replaced with a mixture of synthetic double-stranded oligonucleotides that included 32 codons for residue 71 (codons for all 20 amino acids and the amber codon). The method was nearly 100% efficient for introducing this set of mutations and the observed distribution of codons indicates that the various codons were introduced at equal frequencies.

Class A β-lactamases, which include the pBR322 encoded enzyme, contain a conserved triad (Ser-Thr-x-Lys) at the catalytic site; this site includes Ser 70 whose hydroxyl group opens the β-lactam ring. This study focuses on the conserved Thr 71, whose role in the activity of β-lactamase was previously unknown. Mutants with all 19 possible amino acid substitutions at residue 71 were generated as described above and characterized by the in vivo penam and cephem antibiotic resistance they provided to E. coli LS-1 cells. Surprisingly, cells producing any of 14 of the mutant enzymes display appreciable resistance to ampicillin; only cells with mutants having Tyr, Trp, Asp, Lys, or Arg at residue 71 had no observable resistance to ampicillin. However, all of the mutants are less stable to cellular proteases than the wild type enzyme. These results suggest that Thr 71 is not essential for binding or catalysis but is important for stability of the β-lactamase protein. An apparent change in the substrate specificity of the various mutant enzymes indicates that residue 71 influences the region of the protein that accommodates the side chain attached to the β-lactam ring of the substrate.

To study the role of the naturally occurring disulfide bond in stabilizing β-lactamases that contain mutations at residue 71, a Cys 77 → Ser mutation was introduced into all 19 of these mutants. The resulting doubly mutant proteins were analyzed for their ability to confer resistance to ampicillin in vivo and the relative quantities of the mutant enzymes in whole cell extracts were analyzed by antibody stains of electrophoretic blots. Although the activity and stability of the single mutant Cys 77 → Ser is indistinguishable from the wild type enzyme in physiological conditions, the doubly mutant proteins are much less stable than the analagous single mutants that contain a disulfide bond. Apparently, the presence of the disulfide bond in β-lactamase enables the enzyme partially to overcome instabilities arising from mutations at residue 71. The results presented here also demonstrate that Thr at residue 71 performs a unique and essential role in the stability of β-lactamase.

Thesis Files