title: Engineering a monolignol 4-O-methyltransferase with high selectivity for the condensed lignin precursor coniferyl alcohol.
abstract: Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5 (T133L/E165I/F175I/F166W/H169F), we incrementally remodeled 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. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.
authors.affiliation: From the Biological, Environmental, and Climate Sciences Department, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: Yuanheng
authors.initials: Y
authors.lastname: Cai
authors.affiliation: From the Biological, Environmental, and Climate Sciences Department, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: Mohammad-Wadud
authors.initials: MW
authors.lastname: Bhuiya
authors.affiliation: From the Biological, Environmental, and Climate Sciences Department, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: John
authors.initials: J
authors.lastname: Shanklin
authors.affiliation: From the Biological, Environmental, and Climate Sciences Department, Brookhaven National Laboratory, Upton, New York 11973 cliu@bnl.gov.
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Liu
mesh_terms: D019943:Amino Acid Substitution; D002473:Cell Wall / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D003373:Coumaric Acids; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D008031:Lignin / Q000737:chemistry* / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010636:Phenols / Q000737:chemistry* / Q000378:metabolism; D010666:Phenylpropionates / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D032107:Populus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D011422:Propionates / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D003373:Coumaric Acids; D010636:Phenols; D010666:Phenylpropionates; D010940:Plant Proteins; D011422:Propionates; D011994:Recombinant Proteins; C496130:sinapyl alcohol; D008031:Lignin; C010559:coniferyl alcohol; D008780:Methyltransferases; C495469:p-coumaric acid
keywords: 4-O-methyltransferase; crystal structure; directed evolution; lignin; monolignol; mutagenesis; plant cell wall; protein engineering