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J. Biol. Chem., Vol. 277, Issue 42, 39450-39455, October 18, 2002
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From the Escherichia coli general
NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin
cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed
to bind to it mainly through the isoalloxazine ring. This interaction
was real for riboflavin and FMN, but not for FAD, which bound to Fre
much tighter than FMN or riboflavin. Computer simulations of Fre·FAD
and Fre·FMN complexes showed that FAD adopted an unusual bent
conformation, allowing its ribityl side chain and ADP moiety to form an
additional 3.28 H-bonds on average with amino acid residues located in
the loop connecting F
FAD Is a Preferred Substrate and an Inhibitor of
Escherichia coli General NAD(P)H:Flavin Oxidoreductase*
§,
,**
School of Molecular Biosciences,
Washington State University, Pullman, Washington 99164 and
¶ Department of Chemistry and Chemical Biology, Harvard
University, Cambridge, Massachusetts 02138
5 and F
1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the
overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive
inhibitor with respect to NAD(P)H, decreased the affinity of Fre for
FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The
overlapped binding offers a plausible explanation for the large
Km values of Fre for NADH and NADPH when FAD is the
electron acceptor. Although Fre reduces FMN faster than it
reduces FAD, it preferentially reduces FAD when both FMN and FAD are
present. Our data suggest that FAD is a preferred substrate and an
inhibitor, suppressing the activities of Fre at low NADH concentrations.
*
This work was supported by Grant DE-FG02-00ER62891 from the
NABIR program, Office of Biological and Environmental Research of the United States Department of Energy and by Grant 5-R01-GM61577-03 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Chemistry, University of California,
Berkeley, CA 94720-1460.
**
To whom correspondence should be addressed: School of Molecular
Biosciences, Science Hall 301, Washington State University, Pullman, WA
99164-4234. Tel.: 509-335-2787; Fax: 509-335-1907; E-mail:
xun@mail.wsu.edu.
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