1.8-ANGSTROM 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 replac ement method (MLR) at 1.8 Angstrom resolution with the conventional R factor of 0.187. The crystal structure of FRase I complexed with its c ompetitive inhibitor, dicoumarol, has also been solved at 2.2 Angstrom resolution with the conventional R factor of 0.161. FRase I is a homo dimer, having one FMN cofactor per subunit, which is situated at the i nterface of two subunits. The overall fold can be divided into two dom ains; 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 N ADPH-dependent flavin reductase of Vibrio harveyi (FRP) and the NADH o xidase 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 counter parts 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 su bstrate specificity. Indeed, one of them, Phe124, was found to partici pate in the binding of dicoumarol through stacking to one of the rings of dicoumarol. Upon binding of dicoumarol, most of the exposed re-fac e of the FMN cofactor is buried, which is consistent with the ping pon g bi bi catalytic mechanism. (C) 1998 Academic Press.