FAD Is a Preferred Substrate and an Inhibitor ofEscherichia coli General NAD(P)H:Flavin Oxidoreductase

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

doi:10.1074/jbc.m206339200

Tai Man Louie, Haw Yang + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m206339200

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Abstract

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.

Highlights

Escherichia coli general NAD(P)H:flavin oxidoreductase does not contain any bound flavin cofactor [1, 2]
The most interesting feature is that the loop connecting F␤5 and F␣1 in the flavin-binding domain that normally interacts with the ADP moiety of FAD in other ferredoxin:NADPϩ reductases is exceptionally short in Fre [13]
Results of the Molecular dynamics (MD) simulations (Fig. 4A) showed that the isoalloxazine ring of FAD and the ribityl side chain and the ADP moiety were involved in binding to Fre, offering theoretical support for tighter binding for FAD than for FMN of Rfl

Summary

The abbreviations used are

NAD(P)H:flavin oxidoreductase; Rfl, riboflavin; MD, molecular dynamics; KPi, potassium phosphate; DTT, dithiothreitol; ESI-MS, electrospray ionization mass spectrometry; HPLC, high pressure liquid chromatography. The most interesting feature is that the loop connecting F␤5 and F␣1 in the flavin-binding domain that normally interacts with the ADP moiety of FAD in other ferredoxin:NADPϩ reductases is exceptionally short in Fre [13]. Because the crystal structure of the Fre1⁄7Rfl complex revealed the interactions between the isoalloxazine ring of Rfl and the flavin-binding domain [12] and because the Km values of Fre for Rfl, FMN, and FAD are very similar [2], it has been proposed that all three flavins mainly interact with residues of the flavin-binding domain through the isoalloxazine ring. Studies on the physiological roles of the high affinity of Fre for FAD revealed that FAD is a preferred substrate and inhibitor of Fre. An in vivo model for regulating Fre activities in responding to O2 supply is proposed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION