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.