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

Abstract

We report the structures of an iridoid synthase, nepetalactol synthase, in the presence of NAD+, NADPH or NAD+/10-oxogeranial. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form S-10-oxo-citronellal, a by-product of the enzyme. Tyr178 Oζ is 2.5 Å from the substrate O1, providing the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer leading to formation of the cis-enolate, together with rotation about C4-C5, enabling cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase with almost identical positioning of NADP, Lys146(147), Tyr178(179) and F342(343) but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis.

A multisubstrate reductase from Plantago major: structure-function in the short chain reductase superfamily

“…lanata 24 and the iridoid synthase from C . roseus 25 – 27 . All three enzymes are dimeric in solution as determined by size-exclusion gel purification and the respective crystal structures.…”

Uncoupled activation and cyclisation in catmint reductive terpenoid biosynthesis

“…The enzymatic control of the initial reductive activation step has been structurally characterised in CrISY: crystal structures with cofactor and inhibitor or substrate show binding modes conducive to reduction and formation of an enolate intermediate [21][22][23] . Furthermore, this reduction is stereoselective, as exemplified by the comparison of CrISY, which produces 7S-4a, with AmISY, which produces the enantiomer 18 .…”

Biocatalytic Strategies towards [4+2] Cycloadditions

“…The crystal structures of CrISY co‐crystallised with cofactor and substrate or inhibitor support this: the substrate binds in a linear conformation conducive to reduction but not cyclisation . Investigation into the cyclisation propensity of substrate analogues suggested nepetalactol formation occurs by a stepwise Michael addition, and not Diels–Alder cyclisation (Scheme B); it now appears these experiments were probing not the enzymatic but the general‐acid/buffer catalysed mechanism.…”

“…[39] The crystal structureso fC rISY co-crystallisedw ith cofactor and substrate or inhibitor support this:t he substrate binds in al inear conformationc onducive to reduction butn ot cyclisation. [40][41][42] Investigationi nto the cyclisation propensity of substrate analogues suggested nepetalactol formation occurs by a stepwise Michael addition, and not Diels-Alder cyclisation (Scheme 2B); [43] it now appears these experiments were probing not the enzymatic but the general-acid/buffer catalysed mechanism. Although it has becomec lear that iridoid synthase does not control the cyclisation step, the abundance of certain nepetalactol isomers in some plants led to the hypothesis that stereocontrol may be providedb yasecond enzyme, which will be discussed in the next section.…”