Novel motif is capable of determining CCR and CCR-like proteins based on the divergence of CCRs in plants.

Abstract

Cinnamoyl-coenzyme A reductases (CCRs) have been reported as key enzymes involved in monolignol biosynthesis. In this study, a motif-aware workflow based on a new signature motif effectively distinguished CCRs from CCR-like proteins. The divergence of CCRs and CCR-like sequences in Populus tomentosa Carr, Panicum virgatum L, Oryza sativa L and Selaginella moellendorffii Hieron suggests that NWYCY is not efficient for CCR recognition. The novel motif H202(X)2K205 (CCR-SBM or CCR substrate binding motif) was introduced to distinguish between CCRs and CCR-like proteins. The site-directed mutant R205K in Os(I)CCR-like and H202 in PtoCCR7 resulted in the rescue and loss of activity, respectively, further validating the fact that CCR-SBM is critical for maintaining CCR activity. The molecular docking using feruloyl-cinnamoyl-coenzyme A (CoA) as the ligand and binary PhCCR-NADP structures as receptors indicated an interaction between H202 and K205 with CoA moiety. The genuine CCRs and CCR-like proteins from several angiosperms and gymnosperms were screened using a motif-aware workflow and were validated using a biochemical assay. Our results suggest that the motif-aware workflow is efficient and effective for the identification of CCRs and CCR-like proteins in land plants and can be used as a more accurate way of identifying genuine CCRs among land plants.