1.8Å crystal structure of the major NAD(P)H:FMN oxidoreductase of a bioluminescent bacterium, Vibrio fischeri:: Overall structure, cofactor and substrate-analog binding, and comparison with related flavoproteins

We have solved the crystal structure of FRase I, the major NAD(P)H:FMN oxidoreductase of Vibrio fischeri, by the multiple isomorphous replacement method (MLR) at 1.8 Angstrom resolution with the conventional R factor of 0.187. The crystal structure of FRase I complexed with its competitive inhibitor, dicoumarol, has also been solved at 2.2 Angstrom resolution with the conventional R factor of 0.161. FRase I is a homodimer, having one FMN cofactor per subunit, which is situated at the interface of two subunits. The overall fold can be divided into two domains; 80% of the residues form a rigid core and the remaining, a small flexible domain. The overall core folding is similar to those of an NADPH-dependent flavin reductase of Vibrio harveyi (FRP) and the NADH oxidase of Thermus thermophilus (NOX) in spite of the very low identity in amino acid sequences (10% with FRP and 21% with NOX). 56% of alpha-carbons of FRase I core residues could be superposed onto NOX counterparts with an r.m.s. distance of 1.2 Angstrom. The remaining residues have relatively high B-values and may be essential for defining the substrate specificity. Indeed, one of them, Phe124, was found to participate in the binding of dicoumarol through stacking to one of the rings of dicoumarol. Upon binding of dicoumarol, most of the exposed re-face of the FMN cofactor is buried, which is consistent with the ping pong bi bi catalytic mechanism. (C) 1998 Academic Press.