Mutations in FMN binding pocket diminish chromate reduction rates for Gh-ChrR isolated from Gluconacetobacter hansenii
| Autor: | Yanfeng Zhang, Janin A. Khaleel, Hongjun Jin, Thomas C. Squier, Ruimin Tan, Chunhong Gong |
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| Rok vydání: | 2013 |
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| Zdroj: | Natural Science. :20-24 |
| ISSN: | 2150-4105 2150-4091 |
| DOI: | 10.4236/ns.2013.56a003 |
| Popis: | A putative chromate ion binding site was identified proximal to a rigidly bound FMN from electron densities in the crystal structure of the quinone reductase from Gluconacetobacter hansenii (Gh-ChrR) (3s2y.pdb). To clarify the location of the chromate binding site, and to understand the role of FMN in the NADPH-dependent reduction of chromate, we have expressed and purified four mutant enzymes involving the site-specific substitution of individual side chains within the FMN binding pocket that form non-covalent bonds with the ribityl phosphate (i.e., S15A and R17A in loop 1 between β1 sheet and α1 helix) or the isoalloxanzine ring (E83A or Y84A in loop 4 between the β3 sheet and α4 helix). Mutations that selectively disrupt hydrogen bonds between either the N3 nitrogen on the isoalloxanzine ring (i.e., E83) or the ribitylphos- phoate (i.e., S15) respectively result in 50% or 70% reductions in catalytic rates of chromate reduction. In comparison, mutations that disrupt π-π ring stacking interactions with the isoal-loxanzine ring (i.e., Y84) or a salt bridge with the ribityl phosphate result in 87% and 97% inhibittion. In all cases there are minimal alterations in chromate binding affinities. Collectively, these results support the hypothesis that chromate binds proximal to FMN, and implicate a structural role for FMN positioning for optimal chromate reduction rates. As side chains proximal to the β3/α4 FMN binding loop 4 contribute to both NADH and metal ion binding, we propose a model in which structural changes around the FMN binding pocket couples to both chromate and NADH binding sites. |
| Databáze: | OpenAIRE |
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