Mechanism‐Driven Unlocking of the Activity of Strictosidine Synthase Toward N1‐Substituted Tryptamine

ABSTRACT Strictosidine synthase (STR) catalyzes in nature the enantioselective Pictet–Spengler condensation of secologanin and tryptamine to form ( S )‐strictosidine, a key tetrahydro‐β‐carboline intermediate in the monoterpene indole alkaloid biosynthesis. Extensive studies have revealed that STR exhibits a broad substrate scope, being capable of accepting short‐chain aliphatic and aromatic aldehydes and tryptamine derivatives with substitutions at different carbon positions. However, the activity toward N1‐substituted tryptamine derivatives remained unexplored. To address this gap, in the present study, molecular dynamics simulations and quantum mechanical calculations were performed to identify the reasons responsible for the previously reported inability of STR in catalyzing the reaction of 1‐methyltryptamine with secologanin. It was revealed that this inactivity originates from kinetically prohibitive catalytic steps, caused mainly by the steric clashes in the active site introduced by the N1‐methyl group, rather than substrate binding limitations. Guided by the structural insights, short‐chain aliphatic aldehydes were predicted to alleviate these steric constraints, supported by calculated feasible reaction barriers. Experimental validation confirmed this prediction, enabling the successful asymmetric synthesis of multiple N9‐methyl‐tetrahydro‐β‐carboline derivatives. This work not only advances the fundamental understanding of STR catalysis but also establishes a combined computational‐experimental strategy for exploring and extending the enzyme substrate scope.