Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase

Abstract

Summary The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis‐specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7‐deoxyloganin to loganin (deoxyloganin 7‐hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C. roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C. roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.

Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus)

“…Another 12 ESTs showed some sequence identity to uncharacterized putative CYPs, but none of the 13 CYPs in CrUniGene had exact sequences matches with Catharanthus CYPs in the publicly accessible database. While most of the CYPs in CrUniGene set are quite novel they probably are not candidates for several hydroxylases in MIA biosynthesis such as 7‐deoxyloganic acid 7‐hydroxylase that has yet to be cloned [21].…”

Towards Complete Elucidation of Monoterpene Indole Alkaloid Biosynthesis Pathway

“…As geraniol synthase has been localised to the plastid, geraniol may then be exported to the cytosol where it would be converted to 10-OH geraniol by G10H associated first with vacuolar membranes and later with the endoplasmic reticulum (Guirimand et al, 2009;Madyastha, Ridgway, Dwyer, & Coscia, 1977) and is associated with membrane-bound flavin containing NADPH: cytochrome P450 reductase (CPR) normally required for cytochrome P450 reactions. Very recent studies with IRS also localised expression of this gene to IPAP cells (Geu-Flores et al, 2012), while the second to last (Roepke et al, 2010) and the last steps (Irmler et al, 2000) in secologanin biosynthesis appear to be localised to epidermal cells. Very recent studies with IRS also localised expression of this gene to IPAP cells (Geu-Flores et al, 2012), while the second to last (Roepke et al, 2010) and the last steps (Irmler et al, 2000) in secologanin biosynthesis appear to be localised to epidermal cells.…”

“…Methylation of loganic acid to form loganin is catalysed by loganic acid methyltransferase (LAMT) that has been cloned and characterised from C. roseus . Molecular cloning of SLS (CYP72A1) from C. roseus was followed by its functional expression, and biochemical characterisation in Escherichia coli confirmed its ability to convert loganin into secologanin (Irmler et al, 2000; Fig. This enzyme, first detected in cell suspension culture extracts of Lonicera japonica (Yamamoto, Katano, Ooi, & Inoue, 2000), was suggested to be membrane associated and to be a P450 with requirements for NADPH and O 2 .…”

Camptothecin

“…G10H, which hydroxylates the monoterpenoid geraniol at the C-10 position in the first committed step in the formation of secologanin, is one such cytochrome P450 monooxygenase (Collu et al, 2001). The final reaction for the biosynthesis of secologanin is also catalyzed by a P450 protein, SLS (EC 1.3.3.9; Irmler et al, 2000, Yamamoto, Katano, Ooi, & Inoue, 2000. The enzyme CPR is essential for the activity of G10H and other cytochrome P450 monooxygenases, as it functions in electron transfer from NADPH to cytochrome P450 (Meijer et al, 1993).…”