Enzyme Active Site Design and Combinatorial Mutagenesis for Catalytic Modification in Bacterial Beta-Lactamase

Beta-lactamase is naturally produced in some bacteria to hydrolyze beta-lactam compounds. As a result, these bacteria acquire antibiotic-resistance and become major public health hazards in hospitals and other medical facilities. Extensive research on this enzyme family and related proteins has led to much improvement in defense against bacterial infection. In this study, class C beta-lactamase from Enterobacter cloacae P99 is used as a template for enzyme structure and activity characterization. Based on crystallographic analysis of the P99 enzyme, a group of amino acid residues were identified in the substrate binding pocket (1). They were implicated in contacting the beta-lactam core structure and side groups (2, 3). To further examine the roles of these positions in catalytic turnover, substitutions which permit viable enzyme functions were generated in silico by applying computational modeling methods. To assay biological activities of these customized mutant enzymes, a variant library was created by combinatorial mutagenesis to incorporate the predicted substitutions into target residues in the wild-type template. To do so, DNA sequences encoding the wild-type and mutant enzymes were generated by synthetic gene assembly. These gene sequences were then cloned into expression vector to allow efficient protein production in E. coli BL21 derived host cells. Enzymes were purified by metal affinity column chromatography utilizing the C-terminal 6X histidine tag. Enzymatic activities were quantified for a variety of beta-lactam substrates by in vivo growth assay to determine minimal inhibitory concentrations and by in vitro kinetic analysis (3). All mutants were found active towards hydrolyzing multiple beta-lactam antibiotics in the cephem and penam families including cephalothin and ampicillin. Some mutant enzymes exhibited activities comparable to or higher than the wild-type enzyme, whereas activities decreased in many others. Interestingly, a subset of mutants were found to hydrolyze extended-spectrum cephalosporins more efficiently than the wild-type enzyme. Among them, mutant enzymes containing substitutions in Tyr221 in the omega loop were more active in hydrolyzing cefotaxime and ceftazidime, while their activities towards cephalothin were much reduced. These observations are consistent with previous reports for this family of enzymes, suggesting that conformational changes in the substrate binding pocket play important role in beta-lactam specificity (2, 4). Further characterization of the mutants is underway to quantify their kinetic activities towards cephem and penam antibiotics in relation to structural modification in the active site. Moreover, serine beta-lactamases are known to evolve from penicillin-binding proteins involved in bacterial cell wall synthesis. Some PBPs display transpeptidase activities and share very similar scaffolds as the class C beta-lactamases (5). Thus, the mutants are also being assayed for DD-peptidase activities to assess whether by modifying the active site, these beta-lactamases may acquire peptidase functionality. In the long term, these studies have implications in protein and inhibitor drug design such as with antimicrobial applications.

REFERENCES

1. Lobkovsky, E., Moews, P.C., Liu, H., Zhao, H., Frere, J.-M. and Knox, J.R. (1993) Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase. Proceedings of National Academy of Sciences 90, 11257–11261.  

2. Zhang, Z., Yu, Y., Musser, J.M. and Palzkill, T. (2001) Amino acid sequence determinants of extended spectrum cephalosporin hydrolysis by the class C P99 beta-lactamase. Journal of Biological Chemistry 276, 46568-46574.

3. Goldberg, S. Iannuccilli, W., Nguyen, T., Ju, J. and Cornish, V.W. (2003) Identification of residues critical for catalysis in a class C beta-lactamase. Protein Science 12, 1633-1645.

4. Carter, B.T., Lin, H., Goldberg, S.D., Althoff, E.A., Raushel, J. and Cornish, V.W. (2005) Investigation of the mechanism of resistance to third-generation cephalosporins by class C beta-lactamases by using chemical complementation. ChemBioChem 6, 2055-2067.

5. Massova, I. and Mobashery, S. (1998) Kinship and diversification of bacterial penicillin-binding proteins and beta-lactamases. Antimicrobial Agents and Chemotherapy 42, 1-17.