Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2).
Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.
Chun
Young-Jin
YJ
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Shimada
Tsutomu
T
Sanchez-Ponce
Raymundo
R
Martin
Martha V
MV
Lei
Li
L
Zhao
Bin
B
Kelly
Steven L
SL
Waterman
Michael R
MR
Lamb
David C
DC
Guengerich
F Peter
FP
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D019301:Oleic Acid / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D007850:Lauric Acids; D011994:Recombinant Proteins; D019301:Oleic Acid; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium