Switchgrass PviCAD1: understanding residues important for substrate preferences and activity.
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 in the active site. Mutated proteins displayed lowered or limited activity on cinnamylaldehydes and exhibited altered kinetic properties compared to the WT enzyme, suggesting that key residues important for efficient catalysis had been identified. We have also shown that a sorghum ortholog containing EW, instead of HL in its active site, displayed negligible activity against monolignals. These results indicate that lignifying CADs require a specific set of key residues for efficient activity against monolignals.
USDA-ARS Grain, Forage, and Bioenergy Research Unit, University of Nebraska, 137 Keim Hall, Lincoln, NE 68583-0937, USA.
Aaron J
AJ
Saathoff
Mark S
MS
Hargrove
Eric J
EJ
Haas
Christian M
CM
Tobias
Paul
P
Twigg
Scott
S
Sattler
Gautam
G
Sarath
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D020134:Catalytic Domain; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified; D011487:Protein Conformation; D045868:Sorghum / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000596:Amino Acids; D050505:Mutant Proteins; D008031:Lignin; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase