Mechanism and substrate specificity of the flavin reductase ActVB from Streptomyces coelicolor.
ActVB is the NADH:flavin oxidoreductase participating in the last step of actinorhodin synthesis in Streptomyces coelicolor. It is the prototype of a whole class of flavin reductases with both sequence and functional similarities. The mechanism of reduction of free flavins by ActVB has been studied. Although ActVB was isolated with FMN bound, we have demonstrated that it is not a flavoprotein. Instead, ActVB contains only one flavin binding site, suitable for the flavin reductase activity and with a high affinity for FMN. In addition, ActVB proceeds by an ordered sequential mechanism, where NADH is the first substrate. Whereas ActVB is highly specific for NADH, it is able to catalyze the reduction of a great variety of natural and synthetic flavins, but with K(m) values ranging from 1 microm (FMN) to 69 microm (lumiflavin). We show that both the ribitol-phosphate chain and the isoalloxazine ring contribute to the protein-flavin interaction. Such properties are unique and set the ActVB family apart from the well characterized Fre flavin reductase family.
Filisetti
Laurent
L
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DRDC-CEA/CNRS/ Université Joseph Fourier, 17 Avenue des Martyrs, 38054 Grenoble, Cedex 9, France.
Fontecave
Marc
M
Niviere
Vincent
V
D001665:Binding Sites; D038181:FMN Reductase / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D005415:Flavins; D009711:Nucleotides; D009243:NAD; D005486:Flavin Mononucleotide; D038181:FMN Reductase