Abstract
KslB is one of the few bacterial Pictet-Spenglerases recently identified in the biosynthesis of the β-carboline compound kitasetaline. While previous in vitro studies established that KslB catalyzes the condensation between l-tryptophan and α-ketoglutarate, the reaction mechanism, particularly its stereochemistry, remains poorly understood. This study presents five crystal structures of KslB, capturing key stages of reaction, shedding light on its catalytic dynamics. Among these, alternative binding poses of substrate and reaction product highlighted two significant features: (1) an additional pocket that accommodates l-tryptophan, and (2) two positively charged residues, Lys264 and Arg256, which form salt bridges with the product C1' and C5' carboxylate groups derived from α-ketoglutarate, ensuring a stereoselective process. These structural insights elucidate how KslB governs the stereochemistry of the cyclization process. Accordingly, we propose the configurations for the cyclized intermediate that align with the reaction's stereochemical outcome. Together, these findings offer valuable structural and mechanistic insights into KslB, paving the way for its potential engineering as a Pictet-Spengler biocatalyst.