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

Abstract

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 singlepoint 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.

Design and Optimization for Efficient Production of (S)-Canadine in Escherichia coli

“…It is worth pointing out that, so far, almost all NCSs, including Cj NCS, have been characterized as ( S )-enantiospecific. Only recently, an NCS from Nelumbo nucifera was reported to predominantly function as ( R )-enantiomers . In our efforts to determine the chirality of the target, we conducted fermentation, extraction, and isolation processes, ultimately obtaining purified ( S )-canadine with the predicted negative specific rotation value (Figure S3).…”

“…Only recently, an NCS from Nelumbo nucifera was reported to predominantly function as (R)-enantiomers. 48 In our efforts to determine the chirality of the target, we conducted fermentation, extraction, and isolation processes, ultimately obtaining purified (S)canadine with the predicted negative specific rotation value (Figure S3). Therefore, our study utilized glucose for the production of canadine under normal temperature and pressure conditions without the need for expensive reagents, which is more cost effective and environmentally sustainable.…”

Computational mechanistic investigation of the kinetic resolution of α-methyl-phenylacetaldehyde by norcoclaurine synthase

“…The quantum chemical cluster approach was chosen for investigating the reaction mechanism and exploring the factors controlling the stereospecificity of Tf NCS toward the non-natural aldehyde substrate α-methyl-phenylacetaldehyde. This method has been proven to be powerful in studying the mechanism of enzymatic reactions 11 , 32 – 35 , and elucidating the origins of selectivities 29 , 36 – 38 . All calculations in this work were conducted using Gaussian16 C.01 package with the B3LYP-D3(BJ) density functional method 39 – 43 .…”

“…Norcoclaurine synthases (NCSs) are a subset of PSases that catalyze the PS condensation between dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA), leading to the formation of norcoclaurine, which contains a tetrahydroisoquinoline (THIQ) skeleton. This condensation reaction is the key step in the biosynthesis of BIAs 8 – 11 , making NCSs have received great attention in the designed biosynthetic route of novel alkaloids 12 – 17 . The NCS from Thalictrum flavum ( Tf NCS) stereospecifically catalyzes the PS reaction between dopamine and 4-HPAA to generate ( S )-norcoclaurine (Fig.…”

Chemo-enzymatic synthesis of bioactive compounds from traditional Chinese medicine and medicinal plants

“…This discovery sheds light on the biosynthesis of ( R )-benzylisoquinoline alkaloids for the first time. [20] TcCGT1, the first reported flavone 8- C -glycosyltransferase in plants, is responsible for the biosynthesis of vitexin and orientin in Trollius chinensis . With high catalytic efficiency and adaptability, TcCGT1 directly catalyzes the synthesis of 36 flavone 8- C -glycosides and 44 O -glycosides.…”