Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus

Abstract

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)

Cloning, nucleotide sequence, and transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina thermophila

“…These ubiquitous electron transport proteins participate in a wide variety of redox reactions, and many organisms, including Rhodospirillum rubrum (28), Streptomyces griseolus (15), and Clostridium thermoaceticum (7), contain multiple ferredoxins. Ferredoxins from three different methane-producing organisms have been purified and characterized (9)(10)(11)25); however, the genes encoding these proteins have not been cloned.…”

Electron Transport Pathway for a Streptomyces Cytochrome P450

“…This catalytic activity of P450 105D5 could also be supported by a combination of P. putida PDR and Pdx, which had been used successfully to support oxidative reaction by Streptomyces griseolus P450 by O'Keefe et al (11) or a combination of two flavoproteins, E. coli flavodoxin reductase and Flx (Fig. 2A).…”

“…Chemicals-Streptavidin-peroxidase, 2,2Ј-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, spinach Fdx and FDR, lauric acid, myristic acid, [1-14 C]myristic acid (specific activity 24 mCi mmol Ϫ1 ), palmitic acid, [1-14 C]palmitic acid (specific activity 56 mCi mmol Ϫ1 ), oleic acid, [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleic acid (specific activity 54 mCi mmol Ϫ1 ), linoleic acid, arachidonic acid, [1-14 C]arachidonic acid (specific activity 50 mCi mmol Ϫ1 ), 12-hydroxylauric acid, N,N-diisopropylethylamine, and pentafluorobenzyl bromide were purchased from Sigma. [1-14 C]Lauric acid (specific activity 55 mCi mmol Ϫ1 ) was from GE Healthcare.…”

“…In order to deal with this issue, we first utilized spinach FDR and Fdx, a strategy often employed in work with bacterial systems proposed to involve Fdx proteins (11), realizing that this electron transfer system is neither physiological nor necessarily reflective of kinetic parameters for the natural S. coelicolor systems. Preliminary studies indicated that a combination of commercial spinach FDR and Fdx and purified S. coelicolor P450 105D5 could support the NADPH-catalyzed conversion of oleic acid to a more polar product detected by radio-HPLC ( Fig.…”

“…The streptomycete species examined thus far contain a plethora of Fdx and FDR proteins, and attempts to discern the relevant electron transfer pathways have not been carried out in sufficient detail. Nearly all of the conclusions about the catalytic activities of Streptomyces P450s have come from studies in which electrons are shuttled from heterologous electron transfer components (11), most commonly commercially available spinach FDR and Fdx.…”