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 bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based 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 acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism.
Lei
B
B
Department of Biochemical and Biophysical Sciences, University of Houston, Texas 77204-5934.
Liu
M
M
Huang
S
S
Tu
S C
SC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase; D005486:Flavin Mononucleotide / Q000032:analysis; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017421:Sequence Analysis; D013053:Spectrophotometry; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics*
D003067:Coenzymes; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases