Discovery, Structure, and Mechanism of the (R, S)-Norcoclaurine Synthase for the Chiral Synthesis of Benzylisoquinoline Alkaloids

Benzylisoquinoline alkaloids (BIAs) are key plant metabolites that offer significant pharmacological benefits. While most naturally isolated BIAs are identified as (S)-enantiomers, (R)-configured BIAs are also abundant in specific species. However, the formation mechanism of (R)-enantiospecific BIAs remains largely unknown. Norcoclaurine synthase (NCS)-catalyzed Pictet-Spengler condensation is responsible for the BIAs scaffold formation and establishing a unique chiral carbon center. Nevertheless, all NCSs hitherto identified were strictly (S)-selective. Herein, five NCS homologues in lotus were identified and functionally characterized to be without enantiopreference, namely, being capable of producing (R)-norcoclaurine. We revealed the crystal structure of NnNCS1, one of the five identified NnNCS homologues, showing the enzyme's typical pathogenesis-related protein 10-fold and an active dimeric form. Guided by the mechanistic information from quantum chemical calculations, the single-point mutation of Ile43, Leu60, and Phe101 leads to (R)-enantiospecific mutants. This study unravels the previously obscure pathway of (R)-BIA biosynthesis, thereby providing valuable enzymatic tools for the synthetic biology of (R)-configured BIAs.