Functional and structural studies of isoflavone 4'-O-methyltransferase from Glycyrrhiza glabra L

Flavonoids are a class of plant secondary metabolites. Their O-methylation bond is formed through the catalysis of O-methyltransferases (OMTs), which is a common step in the flavonoid biosynthesis pathway. Plant OMTs are divided into two types: cation-independent type (type 1) and cation-dependent type (type 2). Isoflavone 4'-O-methyltransferase belongs to type 1 OMTs, while anthocyanin O-methyltransferase belongs to type 2 OMTs. Based on previous work, isoflavone 4'-O-methyltransferase (I4'OMT) was identified from Glycyrrhiza glabra L. through in vitro enzyme activity assays. The enzyme is a key enzyme in the biosynthetic pathway of glabridin, which can convert substrate 2,7,4'-trihydroxyisoflavanone into product 2,7-dihydroxy-4'-methoxyisoflavanone. Surprisingly, it was found that it can convert flavanone liquiritigenin into liquiritigenin 4'-methyl ether and liquiritigenin 7-methyl ether in this study. It was indicated that I4'OMT is able to work not only on isoflavone but also flavanone. In addition, anthocyanin O-methyltransferase (VvAOMT) was characterized from Vitis vinifera L. which could convert quercetin to isorhamnetin. In the substrate selectivity study, it was revealed that I4'OMT exhibits activity towards a variety of flavonoid compounds, indicating its broad substrate promiscuity; in contrast, VvAOMT only shows activity towards certain flavonoid compounds, suggesting its substrate specificity. Further, the crystal structures of I4'OMT and VvAOMT were determined through X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, and biochemical assays. The key amino acid residues in I4'OMT/VvAOMT for binding to the substrates liquiritigenin/quercetin were identified and verified (I4'OMT: Y25, F161, D184, Y320, M324; VvAOMT: D151, K154, N178). Furthermore, QM/MM calculations revealed that liquiritigenin maintains structural stability within the catalytic pocket of I4'OMT and adopts an optimal reaction distance to the catalytic center, thereby elucidating the mechanistic basis for I4'OMT-catalyzed methylation at the 4'-OH and 7-OH positions of liquiritigenin. This work not only provides a starting point for a thorough understanding of the biological function of I4'OMT and VvAOMT, but also provides a robust enzymatic tool for biocatalytic synthesis of methylated compounds.