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Mutations in FMN binding pocket diminish chromate reduction rates for Gh-ChrR isolated from Gluconacetobacter hansenii

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DOI: 10.4236/ns.2013.56A003    2,591 Downloads   3,785 Views   Citations

ABSTRACT

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.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Khaleel, J. , Gong, C. , Zhang, Y. , Tan, R. , Squier, T. and Jin, H. (2013) Mutations in FMN binding pocket diminish chromate reduction rates for Gh-ChrR isolated from Gluconacetobacter hansenii. Natural Science, 5, 20-24. doi: 10.4236/ns.2013.56A003.

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