Engineering Monolignol 4-O-Methyltransferases to Modulate Lignin Biosynthesis

Journal Article · Fri Jan 01 00:00:00 EST 2010 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M109.036673· OSTI ID:1019441

Bhuiya, M W; Liu, C

Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily 'plastic' amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States)
• Sponsoring Organization: DOE - OFFICE OF SCIENCE
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 1019441
• Report Number(s): BNL-90651-2010-JA; JBCHA3; R&D Project: BO-147; KC0304000; TRN: US201115%%116
• Journal Information: Journal of Biological Chemistry, Vol. 285, Issue 1; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

09 BIOMASS FUELS
59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ADDITIVES
AFFINITY
AMINO ACIDS
BIOSYNTHESIS
EFFICIENCY
ENZYME ACTIVITY
ENZYMES
IN VITRO
LIGNIN
METHYLATION
MUTAGENESIS
MUTANTS
MUTATIONS
PHENOXY RADICALS
PLASTICS
POLYMERIZATION
POLYMERS
RADICALS
RESIDUES
SPECIFICITY
SUBSTRATES
SYNTHESIS
O-methyltransferase
monolignol
directed evolution
substrate specificity
regio-selectivity