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RAMAN CRYSTALLOGRAPHIC STUDIES OF INHIBITOR REACTIONS IN CLASS A AND D BETA-LACTAMASES
| Title: | RAMAN CRYSTALLOGRAPHIC STUDIES OF INHIBITOR REACTIONS IN CLASS A AND D BETA-LACTAMASES |
| Author: | Totir, Monica Andreea |
| Description: | The issue of inhibitor resistance in Class A beta-lactamase is currently a paramount problem in clinical practice with many patients not responding positively to the antibiotic- beta-lactamase inhibitor combinations. In the clinical context, resistance to drugs such as tazobactam, sulbactam and clavulanic acid often arises by single amino acid replacement in the beta-lactamase. Despite remarkable efforts of various investigators, it is still unclear whether transiently inhibited enzyme is the species that is responsible for the clinical utility of these compounds, or whether irreversible inhibition is required. This work answered this question and elucidated the molecular factors underlying the onset of inhibitor resistance in a Class A beta-lactamase. In this work we have used Raman spectroscopy and Raman microscopy to describe the reaction pathways for tazobactam, sulbactam and clavulanic acid reacting with wild type and several variant forms of SHV-1 beta-lactamase, a Class A beta-lactamase. By comparing the structures and populations of intermediates, for the wild-type enzyme and for clinically relevant resistant forms carrying a replacement amino acid at methionine 69 or serine 130, we have gained unique insight into the changes in molecular mechanisms conferring inhibitor resistance. Raman crystallography was used for the first time to set the conditions necessary to trap a crucial intermediate on the reaction pathway of a Class A beta-lactamase with an inhibitor. These conditions were used by Dr. Focco van den Akker to obtain the X-ray crystallography-derived structures of the inhibitors soaked in different variants of SHV-1 Class A beta-lactamase. In spite of their clinical importance, there is relatively little structural and mechanistic information available about Class D beta-lactamases and these findings are subject to controversy. In this work we have focused on OXA 10 beta-lactamase, a member of the oxacillin cleaving beta-lactamases, and its reaction with various substrates and inhibitors in order to elucidate the structural and mechanistic details of this class. Using the knowledge acquired by studying the reaction of a Class A beta-lactamase with the three clinical inhibitors, we were able to describe why Class D beta-lactamases are poorly inhibited by Class A inhibitors, such as tazobactam and sulbactam. |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=case1171039284
http://hdl.handle.net/2374.OX/16636 |
| Date: | 2007 |
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