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United States Department of Agriculture

Agricultural Research Service


Location: Wheat, Sorghum and Forage Research

Title: Switchgrass PviCAD1: Understanding residues important for substrate preferences and activity

item Saathoff, Aaron
item Hargrove, Mark
item Haas, Eric
item Tobias, Christian
item Twigg, Paul
item Sattler, Scott
item Sarath, Gautam

Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/24/2012
Publication Date: N/A
Citation: N/A

Interpretive Summary: The inability to cheaply and effectively break down plant cell walls is hindering lignocellulosic biofuel development and establishment of full scale biorefineries. Lignin is a complex polymer and one component of plant cell walls that has been identified as negatively impacting conversion of perennial plants, such as switchgrass, into fuel. Lignin biosynthesis is controlled by a suite of enzymes which catalyze specific reactions that ultimately produce the monomeric building blocks (called monolignols) of the lignin polymer. In this study, the protein sequence of switchgrass cinnamyl alcohol dehydrogenase, CAD, an enzyme involved in the final step of generating the monolignols, was altered using site-directed mutagenesis. In this method, specific amino acid residues present in the native (wild-type) enzyme are changed into related or dissimilar amino acids to obtain mutated proteins in order to identify the role of these specific amino acid residues on enzyme function. Switchgrass CAD proteins mutated at specific residues resulted in enzymes with altered activity rates and different substrate preferences when compared to unaltered wild-type CAD. Overall, these data indicated that some residues were critical for the protein to be able to produce monolignols. These data also suggested that other CAD-like proteins present in most plants that lack some of these key residues are unlikely to function in the lignification process. This information could assist plant scientists and others in biofuel research by: (1) demonstrating altered CADs may play a role in developing plants with novel lignins that result in cell walls that are easier to break down; and (2) providing experimental evidence that can be used to differentiate between CADs that are involved in lignification versus other plant functions.

Technical Abstract: Lignin is a major component of plant cell walls and is a complex aromatic heteropolymer. Reducing lignin content improves conversion efficiency into liquid fuels, and enzymes involved in lignin biosynthesis are attractive targets for bioengineering. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis. Although plants contain numerous genes coding for CADs, only one or two CADs appear to have a primary physiological role in lignin biosynthesis. Much of this distinction appears to reside in a few key residues that permit reasonable catalytic rates on monolignal substrates. Here, several mutant proteins were generated using switchgrass wild-type (WT) PviCAD1 as a template to understand the role of some of these key residues, including a proton shuttling HL duo. The selected mutations were designed to alter proton shuttling or substrate binding residues and mimic some of the motifs observed in apparently non-lignifying CADs. Mutated proteins displayed lowered or limited activity on cinnamylaldehydes and exhibited altered kinetic properties compared to the WT enzyme. A sorghum ortholog of a rice CAD containing EW instead HL in its active site displayed negligible activity against monolignals. These results suggest that lignifying CADs require a specific set of key residues for efficient activity against monolignals.

Last Modified: 06/28/2017
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