Novel Biological Catalysts: Mutagenesis of RTEM β-Lactamase to Alter Substrate and Catalytic Specificity
<p>I have used the techniques of site-directed mutagenesis to study the structural requirements for substrate specificity in RTEM-1 β-lactamase and the evolutionary relationship between the β-lactamases and the D,D carboxypeptidases. The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicilli...
| Summary: | <p>I have used the techniques of site-directed mutagenesis to study the structural requirements for substrate specificity in RTEM-1 β-lactamase and the evolutionary relationship between the β-lactamases and the D,D carboxypeptidases. The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A β-lactamases. Both enzymes recognize the β-lactam antibiotics as substrates; however, the β-lactamases have no observable D,D-carboxypeptidase activity.</p>
<p>To investigate the possibility of incorporating D,D-carboxypeptidase activity into [β-lactamase a chimeric protein was prepared by replacing a 28 amino acid sequence of β-lactamase with the corresponding sequence from PBP-5 of E. coli. The resulting chimera was capable of hydrolyzing the D-Ala-D-Ala dipeptide; however, it was determined that a secondary mutation had occurred which inserted a glutamic acid residue between residues 59 and 60.</p>
<p>In Chapter 2, I have performed site-directed mutagenesis on the gene encoding the RTEM-1 PBP-5 chimera to delete the additional residue. The resulting chimera was not thermally stable at 37°C, but is stabilized by interaction with β-lactam compounds such as ampicillin. Despite the finding that the β-lactamase activity was reduced by five orders of magnitude, the chimera displays approximately one percent of the D,D carboxypeptidase activity exhibited by wild-type PBP-5.</p>
<p>In Chapter 3, to further investigate the proposed evolutionary relationship between the PBPs and β-lactamase, I have designed a series of chimeras between the R61 carboxypeptidase/transpeptidase of Streptomyces and RTEM-1 β-lactamase. The design of the chimeras involved building a chimeric substrate binding cavity within the RTEM framework. One chimera (Asp131Asn, Asnl32Phe) was thermally stable and exhibited altered cell morphology in E. coli harboring the chimeric gene.</p>
<p>In Chapter 4, I describe the detailed kinetic analysis of the Asn l32Phe, single-mutant chimera, and the Asp 13 lAsn/Asn l32Phe, double-mutant chimera. The double mutant shows tremendously altered catalytic activity, degrading benzyl penicillin to phenylacetyiglycine and producing the corresponding transpeptidase product, phenylacetyiglycylglycine, when reacted in the presence of glycine. The single mutant, Asnl32Phe, shows no such activity. Furthermore, I have shown that while the Asnl32Phe mutation blocks deacylation in the chimeric proteins, when produced in conjunction with the Aspl3lAsn mutation, the resulting chimera is capable of carboxypeptidase/transpeptidase activity.</p>
<p>Finally, in Appendix I, the description of the site-saturation of RTEM-1 residue 237 is included. Two mutants, Ala237Thr and Ala237Asn, exhibited enhanced preference for cephem over penam antibiotics. The phenotypic screening of all 19 mutants is described.</p> |
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