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purified recombinant MOMT5 and MOMT9 enzyme variants in complex with S-adenosyl-L-homocysteine (SAH), coniferyl alcohol, or sinapyl alcohol, hanging drop vapor diffusion method, crystallization from 22% (w/v) PEG 4000, 0.3 M Mg(NO3)2, pH 7.2, 1 mM DTT, 1 mM SAH, and 1 mM monolignols, X-ray diffraction structure determination and analysis at 1.60-1.73 A resolution, modeling
a promiscuous O-methyltransferase is incrementally evolved to constrain its substrate specificity to guaiacyl lignin precursors. The enzyme with nine amino acid substitutions is functionally specialized for selectively methylating the condensed lignin precursors. Specific remodeling the active site of a monolignol 4-O-methyltransferase creates an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. The engineered promiscuous enzyme MOMT5 is built by remodelling of its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors
a number of monolignol 4-O-methyltransferase variants (MOMTs) are evolved that can trans-methylate the para-hydroxyls of monolignols, which prevents the incorporation of modified precursors into the lignin polymer by disrupting oxidative radical formation. The engineered enzyme is biochemically active against both coniferyl and sinapyl alcohol with similar catalytic turnover rates (e.g. MOMT3 and 4). Therefore, the transgenic plants with overexpressed MOMT maintained similar monomer composition and lignin structure as the wild-type plants