Abstract
A We identified AsKslB from Actinosynnema sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of l-tryptophan and α-ketoglutarate to form kitasetalic acid, a tetrahydro-β-carboline. High-resolution crystal structures of apo, substrate-, intermediate-, and product-bound forms elucidate the full catalytic trajectory and key residues. Crucially, the elusive iminium ion intermediate and a synchronously released water molecule are captured, providing direct structural evidence for the initiating cyclization step of Pictet-Spengler reaction. Glu276 undergoes conformational changes essential for catalysis. These findings offer detailed mechanistic insights into Pictet-Spenglerase function and establish AsKslB as a promising biocatalyst for stereoselective N-heterocycle synthesis.
Supplementary materials
Title
Structural and Functional Insights into the Iminium Ion Intermedi-ate in AsKslB-Mediated Pictet-Spengler Chemistry
Description
Experimental procedures, strains, reagents and primers in-formations, HR-ESI-MS spectrra, protein crystallization and crystallographic elucidation, site-directed mutagenesis, and multiple sequence alignments.
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