Engineering monolignol 4-O-methyltransferases to modulate lignin biosynthesis.

Area of Science:

• Biochemistry and Molecular Biology
• Plant Science
• Enzyme Engineering

Background:

• Lignin, a complex plant polymer, is formed by oxidative coupling of monolignols.
• Native O-methyltransferases methylate lignin precursors only at meta-positions, excluding the para-hydroxyl (4-OH) group.
• The para-hydroxyl is crucial for the oxidative coupling that forms lignin polymers.

Purpose of the Study:

• To engineer a novel O-methyltransferase capable of methylating the para-hydroxyl (4-OH) position of monolignols.
• To investigate the role of specific amino acid residues in enzyme substrate discrimination and regioselectivity.
• To assess the potential of para-methylated monolignols in controlling lignin polymerization.

Main Methods:

• Employed directed evolution and structure-based iterative site-saturation mutagenesis.
• Modified an existing phenylpropene O-methyltransferase.
• Identified and mutated key 'plastic' residues in the enzyme's active site.

Main Results:

• Created a novel monolignol 4-O-methyltransferase by mutating two critical residues.
• Mutations altered substrate specificity and regioselectivity, enabling para-methylation.
• A triple mutant variant exhibited high binding affinity and catalytic efficiency for monolignols, impairing oxidative coupling in vitro.

Conclusions:

• Successfully engineered an enzyme for para-methylation of lignin precursors.
• Demonstrated that para-methoxylation of monolignols inhibits lignin polymerization.
• The novel enzyme holds potential for managing lignin synthesis in plants.