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|Title:||Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles|
Barry III, C.E.
|Source:||Gurumurthy, M., Mukherjee, T., Dowd, C.S., Singh, R., Niyomrattanakit, P., Tay, J.A., Nayyar, A., Lee, Y.S., Cherian, J., Boshoff, H.I., Dick, T., Barry III, C.E., Manjunatha, U.H. (2012-01). Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles. FEBS Journal 279 (1) : 113-125. ScholarBank@NUS Repository. https://doi.org/10.1111/j.1742-4658.2011.08404.x|
|Abstract:||The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F 420) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F 420. The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding. © 2011 FEBS.|
|Source Title:||FEBS Journal|
|Appears in Collections:||Staff Publications|
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