Structural Evidence for the Dopamine-First Mechanism of Norcoclaurine Synthase

Abstract

Norcoclaurine synthase (NCS) is a Pictet-Spenglerase that catalyzes the first key step in plant benzylisoquinoline alkaloid metabolism, a compound family that includes bioactive natural products such as morphine. The enzyme has also shown great potential as a biocatalyst for the formation of chiral isoquinolines. Here we present new high-resolution X-ray crystallography data describing Thalictrum flavum NCS bound to a mechanism-inspired ligand. The structure supports two key features of the NCS “dopamine-first” mechanism: the binding of dopamine catechol to Lys-122 and the position of the carbonyl substrate binding site at the active site entrance. The catalytically vital residue Glu-110 occupies a previously unobserved ligand-bound conformation that may be catalytically significant. The potential roles of inhibitory binding and alternative amino acid conformations in the mechanism have also been revealed. This work significantly advances our understanding of the NCS mechanism and will aid future efforts to engineer the substrate scope and catalytic properties of this useful biocatalyst.

Asymmetric Synthesis of (R)‐1‐Alkyl‐Substituted Tetrahydro‐ß‐carbolines Catalyzed by Strictosidine Synthases

“…Attempts at expressing native sequences of the STRs (Table S1 in the Supporting Information) using various expression plasmids as well as various E. coli hosts, chaperones, and expression conditions resulted in no visible overexpression bands detectable by SDS PAGE, although the enzyme preparations allowed us to run the transformation of the natural substrate to completion within 24 hours at 1–2 m m substrate concentration 6b. Interestingly, highest activities were found when the STRs were expressed with an N‐terminal His 6 ‐tag.…”

Biocatalyzed C−C Bond Formation for the Production of Alkaloids

“…Furthermore, the dual role of Tyr108, contributing to the electrostatic properties of the active site, but also in defining the shape of the cavity entrance was established . Most recently, the studies by Lichman et al . presented new high‐resolution X‐ray crystallography data describing NCS‐ Tf bound to a mechanism‐inspired ligand, which supported the key features of ‘the dopamine‐first mechanism’.…”

Green Routes for the Production of Enantiopure Benzylisoquinoline Alkaloids

“…The first crystallographic structure of NCS from T. flavum in its complex with dopamine and the non-reactive substrate analogue 4-hydroxybenzaldehyde was obtained by our research group [ 70 ] allowing to understand NCS catalytic mechanism and kinetics [ 71 ]. Afterwards, it was demonstrated that a dopamine first mechanism governs the stereoselective production of ( S )-norcoclaurine [ 72 , 73 ] and showed also that the kinetic behavior can explain the ineffectiveness of recombinant NCS in vivo systems. Nevertheless, NCS is a powerful tool for the in vitro asymmetric synthesis of ( S )-norcolaurine and derivatives.…”