VIBRIO-HARVEYI NADPH-FLAVIN OXIDOREDUCTASE - CLONING, SEQUENCING AND OVEREXPRESSION OF THE GENE AND PURIFICATION AND CHARACTERIZATION OF THE CLONED ENZYME

NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacte ria catalyze the reduction of flavin by NAD(P)H and are believed to pr ovide the reduced form of flavin mononucleotide (FMN) for luciferase i n the bioluminescence reaction. By using an oligonucleotide probe base d on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino ac id sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, ap parently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by fo llowing a new and simple procedure involving FMN-agarose chromatograph y as a key step. The same chromatography material was also highly effe ctive in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. D istinct from the free FMN, the bound FMN cofactor showed a diminished A(375) peak and a slightly increased 8-nm red-shifted A(453) peak and was completely or nearly nonfluorescent. The K(m)s for FMN and NADPH a nd the turnover number of this flavin reductase were determined. In co mparison with other flavin reductases and homologous proteins, this fl avin reductase P shows a number of distinct features with respect to p rimary sequence, redox center, and/or kinetic mechanism.