FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

Tai Man Louie; Haw Yang; Pallop Karnchanaphanurach; X Sunney Xie; Luying Xun

doi:10.1074/jbc.M206339200

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FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

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FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

Tai Man Louie, Haw Yang, Pallop Karnchanaphanurach, X Sunney Xie and Luying Xun

The Journal of biological chemistry, Vol.277(42), pp.39450-39455

10/18/2002

DOI: https://doi.org/10.1074/jbc.M206339200

Handle:

https://hdl.handle.net/2376/112653

PMID: 12177066

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Abstract

Flavin-Adenine Dinucleotide - chemistry

Escherichia coli - enzymology

Models, Chemical

Hydrogen

Enzyme Inhibitors - pharmacology

Spectrometry, Fluorescence

Chromatography, High Pressure Liquid

FMN Reductase - antagonists & inhibitors

Dose-Response Relationship, Drug

Flavin-Adenine Dinucleotide - metabolism

FMN Reductase - metabolism

Time Factors

Mass Spectrometry

Escherichia coli - metabolism

Protein Binding

Protein Conformation

Kinetics

Binding Sites

Electrons

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 Fbeta5 and Falpha1 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 K(m) 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.

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Title: FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase
Creators: Tai Man Louie - School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA
Haw Yang
Pallop Karnchanaphanurach
X Sunney Xie
Luying Xun
Publication Details: The Journal of biological chemistry, Vol.277(42), pp.39450-39455
Academic Unit: Molecular Biosciences, School of
Publisher: United States
Grant note: 5-R01-GM61577-03 / NIGMS NIH HHS
Identifiers: 99900547535201842
Language: English
Resource Type: Journal article