The only known biosynthetic route to S-adenosylmethionine (AdoMet) is the reaction of L-methionine and ATP catalyzed by S-adenosylmethionine synthetase (AdoMet synthetase). AdoMet, one of the most versatile metabolites in living cells is involved in methylation reactions, ethylene and polyamine biosynthesis and in radical chemistry. The critical roles of AdoMet have lead to search of inhibitors to selectively inhibit AdoMet synthetase. However, the AdoMet synthetase’s native substrates, L-methionine and ATP are involved in many metabolic processes thus the design of inhibitors that are structural analogs of the substrates remains challenging. Furthermore, the sequence and structure of enzyme particularly in the active site from bacterial to eukaryotic species is highly conserved and thus adds to the difficulty of finding specific and selective inhibitors for pathogenic species. The present study developed a growth curve based assay in E. coli for screening inhibitors against AdoMet synthetase from different bacterial and protozoan species. The complementation assay employs an Escherichia coli metK deletion mutant that makes its growth dependent upon extracellular AdoMet. A plasmid containing either one of the several bacterial, protozoan and mammalian AdoMet synthetase was constructed to complement this E. coli knockout strain. All the tested plasmids were able to rescue the growth of DmetK E. coli strain without exogenous AdoMet. The log phase growth rate is similar for each complemented strain except for longer lag phase for the human MAT1A. For initial proof-of-principle on this growth curve based inhibitor screening, Cycloleucine, a known inhibitor of AdoMet synthetase was used. The growth curve assay showed cycloleucine selectivity to different AdoMet synthetases. Therefore, this approach can be adapted to identifying inhibitors selective to bacterial and protozoan species without exhibiting toxicity against the human enzyme.

Growth based complementation assay in E. coli for inhibitor screening of S-adenosylmethionine synthetase

253rd National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.