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Shi, L., Chen, B., Wang, Z., Elias, D.A., Mayer, M.U., Gorby, Y.A., Ni, S., Lower, B.H., Kennedy, D.W., Wunschel, D.S., Mottaz, H.M., Marshall, M.J., Hill, E.A., Beliaev, A.S., Zachara, J.M., Fredrickson, J.K. and Squier, T.C. (2006) Isolation of a high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1. Journal of Bacteriology, 188, 4705-4714. doi:10.1128/JB.01966-05

has been cited by the following article:

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

  AUTHORS: Janin A. Khaleel, Chunhong Gong, Yanfeng Zhang, Ruimin Tan, Thomas C. Squier, Hongjun Jin

  KEYWORDS: Bioremediation; Chromate Reduction; Enzyme Redesign; Site-Specific Mutagenesis; Chromate Reductase; Flavin Mononucleotide; Promiscuous Enzyme Activities

  JOURNAL NAME: Natural Science, Vol.5 No.6A, June 14, 2013

  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.