Characterization of an 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from Streptomyces griseus.
An 8-hydroxy-5-deazaflavin-dependent oxidoreductase was isolated from the actinomycete Streptomyces griseus and purified 590-fold with 72% overall yield. The enzyme catalyzes electron transfer between 8-hydroxy-5-deazaflavins and NADPH. It seems to be more specific than methanogenic oxidoreductase as it has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate. A molecular weight of 42,000 was found with gel permeation chromatography, while SDS gel electrophoresis indicated the presence of two identical subunits. Maximal enzymatic activity was found at 0.32 M NaCl and pH 5.9 for reduction of 8-hydroxy-5-deazaflavin and pH 7.9 for the reverse reaction. From the kinetic constants it was estimated that the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions. These results indicate the occurrence of 8-hydroxy-5-deazaflavin-dependent electron transfer in microorganisms not belonging to the archaebacteria.
Eker
A P
AP
Biochemical and Biophysical Laboratory, Delft University of Technology, The Netherlands.
Hessels
J K
JK
Meerwaldt
R
R
D002845:Chromatography; D002850:Chromatography, Gel; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005415:Flavins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010777:Photochemistry; D012256:Riboflavin / Q000031:analogs & derivatives*; D013045:Species Specificity; D013305:Streptomyces griseus / Q000201:enzymology*; D013379:Substrate Specificity
D005415:Flavins; C007701:factor 420; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C058515:8-hydroxy-5-deazaflavin-NADPH oxidoreductase; D012256:Riboflavin