Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.
We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Keefe
D P
DP
Central Research and Development Department, E. I. du Pont de Nemours and Company, Inc., Wilmington, Delaware 19880-0402.
Gibson
K J
KJ
Emptage
M H
MH
Lenstra
R
R
Romesser
J A
JA
Litle
P J
PJ
Omer
C A
CA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004790:Enzyme Induction / Q000187:drug effects; D005288:Ferredoxins / Q000235:genetics / Q000302:isolation & purification*; D005798:Genes, Bacterial; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013453:Sulfonylurea Compounds / Q000378:metabolism / Q000494:pharmacology*
D004269:DNA, Bacterial; D005288:Ferredoxins; D013453:Sulfonylurea Compounds; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases