期刊
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
卷 253, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.ijbiomac.2023.126763
关键词
Methyltransferase; Regioselectivity; Crystal structure; Molecular dynamics simulation; Spinosyn
Spinosyn's insecticidal activity depends on the methylation of rhamnose, which is regulated by the substrate specificity of three SAM-dependent methyltransferases. This study elucidated the molecular basis of substrate specificity of SpnK using crystal structures and molecular dynamic simulations, providing insights for engineering spinosyn derivatives.
Rhamnose methylation of spinosyn critical for insecticidal activity is orchestrated by substrate specificity of three S-adenosyl-L-methionine (SAM) dependent methyltransferases (MTs). Previous in vitro enzymatic assays indicate that 3 '-O-MT SpnK accepts the rhamnosylated aglycone (RAGL) and 2 '-O-methylated RAGL as substrates, but does not tolerate the presence of a methoxy moiety at the O-4 ' position of the rhamnose unit. Here we solved the crystal structures of apo and ligand-bound SpnK, and used molecular dynamic (MD) simulations to decipher the molecular basis of substrate specificity. SpnK assembles into a tetramer, with each set of three monomers forming an integrated substrate binding pocket. The MD simulations of SpnK complexed with RAGL or 2 '-O-methylated RAGL revealed that the 4 '-hydroxyl of the rhamnose unit formed a hydrogen bond with a conserved Asp299 of the catalytic center, which is disrupted in structures of SpnK complexed with 4 '-O-methylated RAGL or 2 ',4 '-di-Omethylated RAGL. Comparison with SpnI methylating the C2 '-hydroxyl of RAGL reveals a correlation between a DLQT/DLWT motif and the selectivity of rhamnose O-MTs. Together, our structural and computational results revealed the structural basis of substrate specificity of rhamnose O-MTs and would potentially help the engineering of spinosyn derivatives.
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