Unlike classical P450 enzymes, members of the CYP74 subfamily have atypical reaction mechanisms and require neither oxygen nor a NADPH reductase. CYP74 enzymes are currently divided into four different groups on the basis of their sequence relatedness CYP74A and B include AOS and HPL respectively, showing a strict prefer ence for 13 hydroperoxides, free copy CYP74C includes AOS and HPL which can Inhibitors,Modulators,Libraries convert either 9 and 13 hydroperoxides. Finally, DES are classified as CYP74D. A new nomen clature for CYP74 enzymes, based upon the confirmed substrate and product specificities of recombinant pro teins, has recently been proposed and which assigns CYP74C to only HPLs with dual specificity.

As far as the endocellular distribution of CYP74 members is concerned, Inhibitors,Modulators,Libraries even if a plastidial localisation for AOS and HPL in CYP74A and B groups, respectively is well estab lished, there is very little information on the subcellular localisation of plant HPLs belonging to the CYP74C sub family. Apart from almond seed 9 HPL which is targeted to the endomembrane system and to putative lipid bodies, and two HPLs recently reported from rice targeted to plastids, there is no infor mation Inhibitors,Modulators,Libraries about the localisation of the other HPLs in this subfamily. In contrast to almond 9 HPL which shows a strict preference for 9 hydroperoxides, the other mem bers of the CYP74C subfamily can metabolise both 9 and 13 hydroperoxides and are therefore commonly referred to as 9 13 HPLs. 9 13 HPLs have been reported so far from only a few plant species, namely M. truncatula, melon, cucumber.

In the present work, we have investigated the endocellular localisation of M. truncatula 9 13 HPL, a member of the CYP74C subfamily and its localisation pattern was compared with that of another HPL from M. truncatula Inhibitors,Modulators,Libraries that was predicted from phylogenetic analysis and confirmed through analysis of the recombinant pro tein to be a 13 HPL, a member of the CYP74B subfamily. The link between the unexpected localisation of a member of the CYP74C sub family and the possible activation of the enzyme in vivo is therefore proposed. Results M. truncatula HPLs show different subcellular distributions Inhibitors,Modulators,Libraries Two different cDNA clones from M. truncatula were used in this study the first clone encodes a 9 13 HPL and was produced from mRNA extracted from four week old Rhizobium melitoti inocu lated roots and nodules. the second clone ref 3 encodes a 13 HPL and was produced from mRNA extracted from M. truncatula leaves fed upon by Spodoptera exigua for 24 hours. Similar to other 9 13 HPLs, HPLF was not predicted to contain any canonical chloroplast transit peptide, despite having an unusual pre dicted N terminal sequence enriched with serine and thre onine residues.