Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

Abstract

Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis.

Engineering the biocatalytic selectivity of iridoid production in Saccharomyces cerevisiae

“…The ability of ISY to readily carry out synergistic, cascaded substrate reduction in conjunction with yeast alcohol dehydrogenases suggests that control of substrate oxidation and therefore cyclization must be strictly maintained by the C. roseus GOR, as seen in the in vitro assay. A similar in vitro enzymatic “cross talk” between ISY and a GOR homologue was described previously (Krithika et al, 2015). Future studies into the mechanism of how GOR and ISY coordinate the timing of substrate oxidation and cyclization may lead to new engineering strategies to improve biocatalytic selectivity.…”

Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD+, NADPH, or NAD+/10‐Oxogeranial: Reaction Mechanisms

“…These results confirm the essential nature of Y178 for activity. F342 is also essential and this result is of interest since it supports the role of the equivalent Phe residue (in progesterone reductase) for small enol reductase activity [3b] , while F149 is not essential-consistent with its non-conserved nature. K146 has reduced activity, but no obvious role for this residue is suggested from the X-ray structure.…”

“…K146 has reduced activity, but no obvious role for this residue is suggested from the X-ray structure. It might be involved in the second half reaction, but it is clearly not absolutely essential (as also noted in progesterone reductase by Petersen et al [3e] . A346 and S349, which are close to the substrate, are also not essential for 10-oxogeranial reduction, consistent again with their non-conserved nature.…”

“…In most cases, the reactions proceed via formation of carbo-cationic transition states/reactive intermediates. However, in recent work, an alternative route to the formation of terpenes — involving reductive cyclization—has been reported, for iridoid biosynthesis [3] . Iridoids are monoterpenes that are produced from (C 10 ) 10-oxogeranial ( 1 ) by an NAD(P)H dependent reduction, followed by a cyclization step that involves either a Diels-Alder cycloaddition or a Michael addition [3a, 4] .…”

“…progesterone) they again act as “gatekeepers”, this time impeding access to the active site. Phe>Ala mutations of several PRISEs [3e] favor progesterone reduction, but disfavor small enone reduction. This is consistent with what we see with IRIS in which the F342A mutant is inactive in 10-oxogeranial reduction.…”