biostudies-literatureUnknown11Teng YBPictet-Spengler (PS) reactions are pivotal in the biosynthesis of bioactive alkaloids and pharmaceuticals, yet key structural details underlying their enzymatic catalysis remain insufficiently understood. We identified AsKslB from <i>Actinosynnema</i> sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of l-tryptophan (l-Trp) and α-ketoglutarate (α-KG) to form kitasetalic acid (KA), a tetrahydro-β-carboline (THβC). 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 (IM-1) 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 <i>N</i>-heterocycle synthesis.Synthetic and systems biotechnology247-255https://www.ebi.ac.uk/biostudies/studies/S-EPMC12547825biostudies-literatureStructural and functional insights into the iminium ion intermediate in AsKslB-mediated Pictet-Spengler reaction.PMC12547825Yang XLiu XZou ZChen QXie YZhang XTeng YBQiao ZXie CfalseStructural and functional insights into the iminium ion intermediate in AsKslB-mediated Pictet-Spengler reaction.Pictet-Spengler (PS) reactions are pivotal in the biosynthesis of bioactive alkaloids and pharmaceuticals, yet key structural details underlying their enzymatic catalysis remain insufficiently understood. We identified AsKslB from <i>Actinosynnema</i> sp. ALI-1.44 as a Pictet-Spenglerase with broad substrate scope that catalyzes the stereoselective condensation of l-tryptophan (l-Trp) and α-ketoglutarate (α-KG) to form kitasetalic acid (KA), a tetrahydro-β-carboline (THβC). 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 (IM-1) 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 <i>N</i>-heterocycle synthesis.2026-01-01T00:00:00Z2026 Mar2026-06-05T05:39:31.29Z2026-06-03T03:07:45.74ZS-EPMC125478254114148510.1016/j.synbio.2025.09.017