Abstract
We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We showed here that CYP709C1 is capable of hydroxylating fatty acids at the omega-1 and omega-2 positions. This work was performed after recoding and heterologous expression of a full-length cDNA isolated from a wheat cDNA library in an engineered yeast strain. Investigation on substrate specificity indicates that CYP709C1 metabolizes different fatty acids varying in their chain length (C12 to C18) and unsaturation. CYP709C1 is the first identified plant cytochrome P450 that can catalyze sub-terminal hydroxylation of C18 fatty acids. cis-9,10-Epoxystearic acid is metabolized with the highest efficiency, i.e. K((m)(app)) of 8 microM and V(max(app)) of 328 nmol/min/nmol P450. This, together with the fact that wheat possesses a microsomal peroxygenase able to synthesize this compound from oleic acid, strongly suggests that it is a physiological substrate. Hydroxylated fatty acids are implicated in plant defense events. We postulated that CYP709C1 could be involved in plant defense by producing such compounds. This receives support from the observation that (i) sub-terminal hydroxylation of 9,10-epoxystearic acid is induced (15-fold after 3 h) in microsomes of wheat seedlings treated with the stress hormone methyl jasmonate and (ii) CYP709C1 is enhanced at the transcriptional level by this treatment. CYP709C1 transcript also accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital. This indicates a possible detoxifying function for CYP709C1 that we discussed.
Highlights
We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes
Cloning of CYP709C1—In an attempt to clone all cytochromes P450 from wheat involved in xenobiotic metabolism, the screening of a cDNA library from wheat treated with cloquintocet-mexyl and phenobarbital led us to isolate and sequence a full-length cDNA of 1975 bp containing an open reading frame of 1545 bp
Sequence alignment showed that the deduced protein was a new member of the CYP709 family of cytochrome P450 named CYP709C1
Summary
We cloned and characterized CYP709C1, a new plant cytochrome P450 belonging to the P450 family, that so far has no identified function except for clustering with a fatty acid metabolizing clade of P450 enzymes. We postulated that CYP709C1 could be involved in plant defense by producing such compounds Functional expression in an engineered yeast allowed us to show that CYP709C1 metabolizes fatty acids, and among all the fatty acids tested, cis-9,10-epoxystearic acid produced by a peroxygenase is hydroxylated at positions -1 and -2 with the highest efficiency This is the first report of a sub-terminal hydroxylase of long chain fatty acid cloned in plant. CYP709C1 is up-regulated at the transcriptional level by a treatment of wheat seedlings with a combination of the safener naphthalic acid anhydride with phenobarbital or by the stress hormone methyl jasmonate. Its physiological meaning remains to be established, and we discuss its possible implication in the plant defense and detoxifying process
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