Cloning, Functional Expression, and Characterization of CYP709C1, the First Sub-terminal Hydroxylase of Long Chain Fatty Acid in Plants

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 -1 and -2 positions. This work was performed after recoding and heterologous expression of a fulllength 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 M 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.

CYP704B1 Is a Long-Chain Fatty Acidω-Hydroxylase Essential for Sporopollenin Synthesis in Pollen of Arabidopsis

“…This makes the comparison of enzymes characterized from different plants difficult. Nevertheless, the level of activity measured with CYP704B1 is similar to the activities of previously described fatty acid hydroxylases expressed using the same heterologous system and assayed using the same experimental procedure Kandel et al, 2005Kandel et al, , 2007. It is to be noted, however, that contrary to the fatty acid hydroxylases CYP94A1 from Vicia sativa, CYP709C1 from wheat (Triticum aestivum), and CYP94C1 from Arabidopsis (Pinot et al, 1999;Kandel et al, 2005Kandel et al, , 2007, CYP704B1 does not metabolize 9,10-epoxystearic acid with a much higher efficiency than other fatty acids tested.…”

The Cytochrome P450 Enzyme CYP96A15 Is the Midchain Alkane Hydroxylase Responsible for Formation of Secondary Alcohols and Ketones in Stem Cuticular Wax of Arabidopsis

“…This distinguishes it from most other known P450 enzymes, alkane hydroxylases, and fatty acid hydroxylases, which can only catalyze reactions on terminal or subterminal carbons of the substrates (e.g. v-1 or v-2; Whyte et al, 1998;van Beilen et al, 2003;Kandel et al, 2005Kandel et al, , 2006. To date, only one other P450 enzyme with in-chain hydroxylase activity has been described and it prefers C12 fatty acid substrates very different from the VLC alkane substrates of MAH1 (Morant et al, 2007).…”

Metabolomics for the characterization of cytochromes P450-dependent fatty acid hydroxylation reactions in Arabidopsis

“…CYP709C1 can hydroxylate fatty acids at the w-1 and w-2 positions. CYP709C1 transcripts accumulated after treatment with a combination of the safener naphthalic acid anhydride and phenobarbital, indicating a possible detoxifying function for CYP709C1 (Kandel et al 2005). CYP78A1 from maize has also been reported to be a lauric acid whydroxylase, and CYP703A2 in Arabidopsis catalyzes the in-chain hydroxylation of lauric acid during sporopollenin biosynthesis (Morant et al 2007).…”