Strictosidine Synthase:  Mechanism of a Pictet−Spengler Catalyzing Enzyme

Abstract

The Pictet-Spengler reaction, which yields either a β-carboline or a tetrahydroquinoline product from an aromatic amine and an aldehyde, is widely utilized in plant alkaloid biosynthesis. Here we deconvolute the role that the biosynthetic enzyme strictosidine synthase plays in catalyzing the stereoselective synthesis of a β-carboline product. Notably, the rate-controlling step of the enzyme mechanism, as identified by the appearance of a primary kinetic isotope effect (KIE), is the rearomatization of a positively charged intermediate. The KIE of a nonenzymatic Pictet-Spengler reaction indicates that rearomatization is also rate-controlling in solution, suggesting that the enzyme does not significantly change the mechanism of the reaction. Additionally, the pH dependence of the solution and enzymatic reactions provides evidence for a sequence of acid-base catalysis steps that catalyze the Pictet-Spengler reaction. An additional acid-catalyzed step, most likely protonation of a carbinolamine intermediate, is also significantly rate controlling. We propose that this step is efficiently catalyzed by the enzyme. Structural analysis of a bisubstrate inhibitor bound to the enzyme suggests that the active site is exquisitely tuned to correctly orient the iminium intermediate for productive cyclization to form the diastereoselective product. Furthermore, ab initio calculations suggest the structures of possible productive transition states involved in the mechanism. Importantly, these calculations suggest that a spiroindolenine intermediate, often invoked in the Pictet-Spengler mechanism, does not occur. A detailed mechanism for enzymatic catalysis of the β-carboline product is proposed from these data.

A Pictet-Spengler ligation for protein chemical modification

“…1B), we were guided by a thorough kinetic study of the Pictet-Spengler reaction indicating that formation of an iminium ion intermediate is partially rate-limiting (34). Taking note of the rapid rates at which α-effect amines condense with aldehydes and ketones (35), we hypothesized that the rate of iminium ion formation could be enhanced by replacing the aliphatic amine of tryptamine with an aminooxy moiety.…”

Enzymatic condensation of dopamine and acetaldehyde: a salsolinol synthase from rat brain

“…It will be of great interest to compare the sequence of salsolinol synthase with other two PictetSpengler-ases, such as strictosidine synthase and norcoclaurine synthase (Luk et al 2007;Maresh et al 2008). This study is currently under investigation in our laboratory.…”

The Pictet–Spengler Reaction in Nature and in Organic Chemistry

“…The isotope effect measured indicates that all the reaction steps before rearomatization by deprotonation are reversible. [50,51] In principal, the nucleophilic C3 atom, instead of C2, could also attack the iminium carbon atom to perform the ring closure, but this would then lead to a spirocyclic intermediate followed by a rearrangement and formation of a six-membered ring. Ab initio calculations on the transitionstate energy of such a spiro intermediate and rearrangement from a five-to a six-membered ring system show the process to be energetically unfavorable.…”