CYP83B1 Is the Oxime-metabolizing Enzyme in the Glucosinolate Pathway in Arabidopsis

Abstract

and the ‡ ‡IACR-Rothamsted, Harpenden, AL5 2JQ, United Kingdom CYP83B1 from Arabidopsis thaliana has been identified as the oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates. Biosynthetically active microsomes isolated from Sinapis alba converted p-hydroxyphenylacetaldoxime and cysteine into S-alkylated p-hydroxyphenylacetothiohydroximate, S-(p-hydroxyphenylacetohydroximoyl)-L-cysteine, the next proposed intermediate in the glucosinolate pathway. The production was shown to be dependent on a cytochrome P450 monooxygenase. We searched the genome of A. thaliana for homologues of CYP71E1 (P450ox), the only known oxime-metabolizing enzyme in the biosynthetic pathway of the evolutionarily related cyanogenic glucosides. By a combined use of bioinformatics, published expression data, and knock-out phenotypes, we identified the cytochrome P450 CYP83B1 as the oxime-metabolizing enzyme in the glucosinolate pathway as evidenced by characterization of the recombinant protein expressed in Escherichia coli. The data are consistent with the hypothesis that the oxime-metabolizing enzyme in the cyanogenic pathway (P450ox) was mutated into a "P450mox" that converted oximes into toxic compounds that the plant detoxified into glucosinolates.Glucosinolates are naturally occurring amino acid-derived S-glucosides of thiohydroximate-O-sulfonates. They co-occur with endogenous thioglucosidases called myrosinases that upon tissue damage hydrolyze glucosinolates into a wide range of degradation products such as e.g. isothiocyanates, nitriles, and thiocyanates. Glucosinolates (or rather their degradation products) are involved in plant defense and constitute characteristic flavor compounds and cancer-preventive agents in Brassica vegetables.The biosynthetic pathway from precursor amino acid to the core glucosinolate structure has been well studied, and many of the intermediates are known, including oximes, thiohydroximic acids, and desulfoglucosinolates (1, 2). Recently, it has been shown that cytochromes P450 belonging to the CYP79 family catalyze the conversion of amino acids to oximes (3-7). Little is known about the formation of thiohydroximic acids from oximes. The remaining part of the pathway for the core structure involves a UDP-glucose:thiohydroximic acid glucosyltransferase and a sulfotransferase (for review, see Ref.2).It has been proposed that aci-nitro compounds are intermediates in the conversion of oximes to thiohydroximic acids (8). This was supported by isolation of 1-nitro-2-phenylethane from Tropaeolum majus shoots and by in vivo conversion of phenylacetaldoxime into 1-nitro-2-phenylethane and of 1-nitro-2-[1,2-14 C]phenylethane into benzylglucosinolate (9). The aci-nitro is proposed to be conjugated with a sulfur donor to produce an S-alkyl thiohydroximate, possibly by a glutathione S-transferase (2). Biochemical studies indicate that the S-alkyl thiohydroximate is subsequently hydrolyzed to the thiohydroximic acid by a C-S lyase (10).Glucosinolates are related to cyanogenic glucosides because both...

Camalexin is synthesized from indole-3-acetaldoxime, a key branching point between primary and secondary metabolism in Arabidopsis

“…Because CYP83B1 has a very high affinity for IAOx (14), this observation suggests that the IAOx-metabolizing enzymes in camalexin and IAA biosynthesis compete with CYP83B1 for IAOx, unless independent pools exist. Interestingly, S-alkyl thiohydroximate intermediates that are produced in vitro by the oximemetabolizing CYP83 enzymes in the glucosinolate pathway undergo internal cyclization to form a thiazoline ring with structural similarity to the thiazol ring in camalexin (15). CYP83A1 and CYP83B1 belong phylogenetically to the CYP71 family (31), as does the oxime-metabolizing enzyme in the biosynthesis of cyanogenic glucosides (32).…”

“…In Arabidopsis, CYP79B2 (12,13) and CYP79B3 (12) catalyze the conversion of tryptophan into indole-3-acetaldoxime (IAOx) that is channeled by the oxime-metabolizing CYP83B1 into the biosynthetic pathway of indole glucosinolates (14,15). Furthermore, IAOx has been shown to be a precursor of the phytohormone indole-3-acetic acid (IAA) as evidenced by enzyme studies with radiolabeled IAOx (16) and the high-auxin phenotype of knockout mutants in postoxime enzymes in glucosinolate biosynthesis, such as sur1 (17) and sur2 (14,18).…”

Nitrile Pathway Involving Acyl-CoA Synthetase

“…In glucosinolateproducing plants (e.g. Arabidopsis), aldoximes are formed from amino acids by cytochrome P450 belonging to the CYP79 family (52)(53)(54)(55). We are interested in the biosynthesis of a substrate (of aldoxime dehydratase), which remains undetermined.…”

A New Member of Plant CS-lyases

“…The identification of a new plant CS-lyase member, the first plant cystine lyase identified at a molecular level, adds impetus to a number of potential investigations such as the possible involvement of CS-lyases in glucosinolate biosynthesis (2,35), indole acetic acid metabolism, and sulfur metabolism. The inducible nature of CORI3 also makes it highly desirable to find out more about the biological role of cystine lyases in plants and CORI3 in particular.…”

“…The putative gene sequence of several alleles of the locus in question was obtained (13), but the catalytic function of the protein encoded by rooty/superroot1 (rooty/superroot1) remains unknown despite a multitude of approaches (13,14). If rooty/superroot1 acts as a CS-lyase in planta, as suggested by the sequence similarity of rooty/superroot1 and CORI3, one possible role could be as the proposed CS-lyase that catalyzes the desulfhydration of S-alkyl thiohydroximate to afford thiohydroximate in the glucosinolate biosynthetic pathway (2,35), with an indirect effect upon indole acetic acid accumulation in a manner examplified by CYP83B1 (34).…”