Structural and Enzymatic Characterization of the Sorghum Lignin Biosynthesis Pathway

Alexander M. Walker

Production of second-generation, or cellulosic, biofuels is complicated significantly by the extensive lignification of plant cellulosic materials. Physiologically, lignins form chemical bonds with hemicelluloses and celluloses and thereby establish networks between the cellulose microfibrils in the plant cell wall and thereby preventing the activity of cellulolytic enzymes required for saccharification to produce sugars utilizable for microbial fermentation. The mature lignin chain arises primarily by the radical-coupling of p-coumaryl, coniferyl, and sinapyl alcohols, which are synthesized by the monolignol biosynthesis pathway and give rise to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units in the mature lignin polymer, respectively. While silencing and downregulation of genes involved in monolignol biosynthesis have resulted in severe dwarfism phenotypes, control of the monolignol biosynthesis pathway using enzyme-engineering approaches provides reduction of plant lignification without adversely impacting plant growth, proving an attractive option to engineer crops to complement industrial-scale biofuel production. This dissertation is composed of two studies that investigate enzymes from the Sorghum bicolor monolignol biosynthesis pathway. S. bicolor has superior saccharification and lower nutrient requirements relative to competitor second-generation biofuel crops, increasing bioethanol yields while decreasing the cost associated with its agricultural production. The first study is a structural and enzymatic characterization of a hydroxycinnamoyltransferase (HCT) from S. bicolor. HCT is a member of the BAHD family of CoA-dependent acyltransferases and is representative of a broad functional class of plant acyltransferases. We determined the crystal structure of HCT, identified critical residues for catalysis and substrate-binding, and proposed a mechanism for HCT catalysis. In combination, this knowledge guided the construction of mutants that might give rise to favorable bioenergy traits in vivo. The second study investigated the S. bicolor caffeoyl-CoA O-methyltransferase (CCoAOMT), a SAM-dependent and cation-dependent type-2 plant O-methyltransferase that catalyzes the methylation of a cinnamoyl-CoA thioester. We determined the structure of the SbCCoAOMT-SAM binary complex and provide biophysical data clarifying SbCCoAOMT substrate and cation preference. We identified a novel binding pose for the thioester substrate using molecular docking and performed site-directed mutagenesis to establish a general catalytic mechanism for CCoAOMT enzymes.

Structural and Enzymatic Characterization of the Sorghum Lignin Biosynthesis Pathway

Files and links (1)

Abstract

Metrics

Details