Steven A. Sattler, Alexander M. Walker, Wilfred Vermerris, Scott E. Sattler, ChulHee Kang

Cinnamoyl-CoA reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-CoA esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reducases from Sorghum bicolor, a strategic plant for the bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues Thr154 and Tyr310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Thr154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative SbCCR genes were discovered from sorghum genomic data. One of these, SbCCR3 displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could be indicative of playing a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge of critical residues on their substrate preference among CCRs, and provide the first three-dimensional structure information for a CCR from a monocot species.