Studies of the Roles of Residues 71 and 72 of RTEM-1 Beta-Lactamase and the Structure-Function Relationships between Beta-Lactamases and D,D-Carboxypeptidases by Mutagenesis

Citation

Chang, Yie-Hwa (1987) Studies of the Roles of Residues 71 and 72 of RTEM-1 Beta-Lactamase and the Structure-Function Relationships between Beta-Lactamases and D,D-Carboxypeptidases by Mutagenesis. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/k85q-w423. https://resolver.caltech.edu/CaltechTHESIS:08302019-140132981

Abstract

The technique of site-directed mutagenesis, including oligonucleotide-directed mutagenesis and cassette mutagenesis, enables us to make any possible structural alterations at desired sites in proteins whose genes have been cloned. This technique when combined with biochemical and X-ray crystallographic analysis has been proved to be a very powerful tool for studying the structure-function relationships in proteins. Using this technique, I have studied some functional requirements of RTEM-1 betalactamase and the structure-function relationships between beta-lactamases and D,D-carboxypeptidases.

Beta-lactamases can catalyze the hydrolysis of the amide bond in the beta-lactam ring of penam and cephem antibiotics. The precise catalytic mechanism of these enzymes is still unclear; the specific amino acid residues, in addition to the active site Ser, involved in binding or catalysis remain unknown.

In Chapter I, to study the role of a conserved residue 71, Thr, of RTEM-1 beta-lactamase, I have changed this residue into Ile, Leu and Met, respectively, using oligonucleotide-directed mutagenesis. The results indicate that although the Thr residue may not be directly involved in binding or catalysis, both the methyl and the hydroxyl group on the beta-carbon of Thr71 play a very important role in stabilizing the conformation of the wild-type RTEM-1 beta-lactamase; in this regard, the methyl group seems more important than the hydroxyl group.

It has been proposed that beta-lactamases may have evolved from D,D-carboxypeptidases. Recently, structural data became available for comparing a penicillin-binding-protein (or a D,D-carboxypeptidase) from Streptomyces R61 with class A beta-lactamases from B. licheniformis 749/C and B. cereus 561. The significant similarity found by X-ray crystallography in the spatial arrangement of the elements of secondary structure in these proteins strongly supports the hypothesis described above. Interestingly, there are conserved triads for both class A beta-lactamases and D,D-carboxypeptidases in the immediate vicinity of the active site Ser; the one for class A beta-lactamases is Ser-Thr-Xaa-Lys; however, the one for D,D-carboxypeptidases is Ser-Xaa-Thr-Lys.

In Chapter II, to further study the roles of residues 71 and 72 of RTEM-1 beta-lactamases and to study the possibility of creating a substantial D,D-carboxypeptidase activity within the background of beta-lactamase structure, I have changed the diad Thr71Phe72 of RTEM-1 beta-lactamase into Thr71thr72 and the D,D-carboxypeptidase-like sequences, Leu71Thr72 and Ile71Thr72. The results indicate that although these two residues may not be directly involved in binding or catalysis, they may play a very important role in keeping the active site Ser and the conserved Lys residue in the correct orientation for efficiently catalyzing the hydrolysis of the beta-lactam antibiotics by beta-lactamases. Moreover, none of these mutant beta-lactamases shows detectable D,D-carboxypeptidase activity, suggesting that I may have to change more than two amino acid residues around the active site Ser of RTEM-1 beta-lactamase to generate a mutant beta-lactamase which can catalyze appreciable D,D-carboxypeptidase activity.

In Chapter III, to further study the structure-function relationships between beta-lactamases and D,Dcarboxypeptidases, I constructed a hybrid protein by the replacement of a polypeptide chain containing 29 amino acid residues (from residue 47 to 75, in the number system of Ambler) of RTEM-1 beta-lactamase with the corresponding sequence containing 30 amino acid residues of PBP-5 of E. coli. The results indicate that beta-lactam antibiotics can not only induce the catalytic activity of this hybrid protein but also stabilize the conformation of this protein. The purified hybrid protein shows about 10^-3 of the specific activity of the wild-type beta-lactamase against benzylpenicillin and, interestingly, given the objective, this hybrid protein also shows about 1.8% of the D,D-carboxypeptidase activity of the wild-type PBP5 of E. coli. Furthermore, this hybrid protein shows no detectable transpeptidase activity, suggesting that the alpha-G helix in D,D-carboxypeptidase may play an important role in the transpeptidation reaction.

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