Hydroperoxide bicyclase CYP50918A1 of Plasmodiophora brassicae (Rhizaria, SAR): Detection of novel enzyme of oxylipin biosynthesis

The genome of the cabbage clubroot pathogen Plasmodiophora brassicae Woronin 1877 (Cercozoa, Rhizaria, SAR), possesses two expressed genes encoding the P450s that are phylogenetically related to the enzymes of oxylipin biosynthesis of the CYP74 clan. The cDNA of one of these genes (CYP50918A1) has been expressed in E. coli. The preferred substrate for the recombinant protein, the 13-hydroperoxide of alpha-Tinolenic acid (13-HPOT), was converted to the novel hetembicyclic oxylipins, plasmodiophorols A and B (1 and 2) at the ratio ca. 12:1. Compounds 1 and 2 were identified as the substituted 6-oxabicyclo[3.1.0]hexane and 2-oxabicyclo[2.2.1]heptane (respectively) using the MS and NMR spectroscopy, as well as the chemical treatments. The O-18 labelling experiments revealed the incorporation of a single O-18 atom from [O-18(2)]13-HPOT into the epoxide and ether functions of products 1 and 2 (respectively), but not into their OH groups. In contrast, the O-18 from [O-18(2)]water was incorporated only into the hydroxyl functions. One more minor polar product, plasmodiophorol C (3), identified as the cyclopentanediol, was formed through the hydrolysis of compounds 1 and 2. Plasmodiophomls A-C are the congeners of egregiachlorides, hybridalactone, ecklonialactones and related bicyclic oxylipins detected before in some brown and red algae. The mechanism of 13-HPOT conversions to plasmodiophorols A and B involving the epoxyallylic cation intermediate is proposed. The hydmperoxide bicyclase CYP50918A1 is the first enzyme controlling this kind of fatty acid hydmperoxide conversion.

Automated summary

Genomic analysis of the cabbage clubroot pathogen revealed the presence of two expressed genes encoding P450 enzymes related to oxylipin biosynthesis. The recombinant protein from one of these genes successfully converted a specific substrate into novel oxylipins, providing insights into the conversion mechanism. This study sheds light on the role of the enzyme CYP50918A1 in controlling fatty acid hydroperoxide conversion and the formation of unique oxylipins in this pathogen.