Structural insights into differential substrate regioselectivity and cofactor-substrate cooperativity between SOMT and COMT

Abstract

Methyltransferase typically refers to the enzymes that catalyse methyl group transfer from S-adenosylmethionine (SAM) cofactor to substrate. Methyltransferases target a broad spectrum of substrates and reactive sites. The transfer of a small methyl group can profoundly alter physiological activity. Industrially, methyltransferases promise a green route for versatile R-group transfer and late-stage modifications of complex chemicals. SOMT and COMT are two phylogenetically related O-methyltransferases from Sorghum bicolor that show differential substrate regioselectivity and cofactor-substrate cooperativity. This study aimed to investigate the structural basis of the differential substrate regioselectivity and cofactor-substrate cooperativity of SOMT and COMT, and to implement the uncovered insights out of SOMT and COMT context. The collaborative works in this study demonstrate that SOMT and COMT shares similar binding affinity profile towards resveratrol (substrate) and pterostilbene (product) but COMT exhibits poorer methylating activity. Through X-ray crystallography, SOMT-resveratrol-β-NAD ternary complex was solved at 1.72 Å in this study to uncover the structural basis of resveratrol binding to SOMT. The binding basis of resveratrol is driven by the hydrophobic interactions between the pocket’s residues and the resveratrol’s stilbene backbone and direct hydrogen bond between catalytic histidine and resveratrol reactive site, 3-OH. Conversely, docking with COMT showed resveratrol binding orientations perturbs between non-productive state and productive state at similar free binding energy. In complementary to the structural insights, mutagenesis study showed that the mutants, SOMTF337N and COMTN323F, exchanged the activity upon one single mutation. This finding evidently determines F337 and N323 as the sole regioselectivity governing residue corresponding to the differential resveratrol methylating activity. To explain this observed regioselectivity, the term “polarity pairing” was introduced to define the polar-to-polar and non-polar-to-non-polar pairing between the enzyme and substrate. Secondly, cofactor-substrate cooperativity characterization of SOMT revealed a strong cofactor-dependent positive substrate cooperativity, whose triggering factor was traced to the adenosine functional group. Structural comparison of apo SOMT and ternary complex revealed the association of closure of SAM-binding domain and rearrangement of Trp279 with resveratrol binding. Integrating structural dynamics insights from different bound states and different homologs further suggest that the Trp279 π-stacking network as a spontaneous epistasis gain. Collectively, this study proposes the mechanism of cofactor-dependent SOMT positive cooperativity is driven by the closure of SAM-binding domain which subsequently confines the pocket and forces Trp279 and His196 to rearrange to form a π-stacking network to promote substrate binding. Meanwhile, this study demonstrated that SOMT-resveratrol-β-NAD co-crystallization can be faithfully reproduced by replacing β-NAD with some adenine-moiety harboring cofactors such as the cellular abundance ATP and resveratrol precursor p-coumaroyl CoA; suggesting a plausible orthostery in SOMT. Lastly, this study employed structure alignment to demonstrate the wide occurrence of polarity pairing among the homologous methyltransferases. In conclusion, this study provides a structural basis of polarity pairing regioselectivity of SOMT and COMT; proposes a structural basis of novel cofactor-dependent positive substrate cooperativity of SOMT and shows structural evidence of the wide occurrence of polarity pairing among homologous methyltransferases. The findings will foster the development of methyltransferases biocatalyst in multiple ways.