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Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR

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Abstract

Main conclusion

Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants.

Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.

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Acknowledgments

This work was jointly supported by the National Natural Science Foundation [NSF 31300498 to Y.G.], High Technology Research and Development 863 Program [2011AA100203 to X.N.JIANG.], [Grant Numbers J1103516, J1310005, ITR 13047] the Basic Science Basement Facility Buildup and Talent Training Program Project from National Natural Science Foundation of China (NSFC) and PCSIRT, and the Fundamental Research Funds for the Central Universities [BLYJ201504 to N.C.].

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Authors and Affiliations

  1. College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China

    Nan Chao, Ning Li, Qi Qi, Shuang Li, Tong Lv, Xiang-Ning Jiang & Ying Gai

  2. The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration, Beijing, 100083, China

    Xiang-Ning Jiang & Ying Gai

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  1. Nan Chao
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Correspondence to Ying Gai.

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Online Resource 1 Primers used for cloning PtoCCR and PtoCCR-like genes from P. tomentosa. Online Resource 2 Putative CCR proteins used for phylogenetic analysis. Online Resource 3 Conditions used for HPLC–MS to identify hydroxycinnamoyl-coA esters. Online Resource 4 Homology matrix of 12 sequences (DNAman). Online Resource 5 Identification of chemically synthesized hydroxycinnamoyl-CoA esters by HPLC–MS. (A–D) Chromatographs of caffeoyl-CoA, feruloyl-CoA, p-coumaroyl-CoA, and sinapoyl-CoA, respectively. (E–H) Mass spectra of caffeoyl-CoA, feruloyl-CoA, p-coumaroyl-CoA, and sinapoyl-CoA, respectively. Online Resource 6 Summary of active sites in CCRs and corresponding amino acids in 11 PtoCCR and PtoCCR-like proteins. Online Resource 7 Expression profiles of 11 PtoCCR and PtoCCR-like genes in different tissues from P. tomentosa based on microarray datasets. The transcript abundance is indicated by the blue–red gradient, as shown in the legend. Tissues or specific parts of plants are indicated. Online Resource 8 Phylogenetic trees of CCRs from core dicots. (A) Phylogenetic tree of CCRs from eurosids II. (B) Phylogenetic tree of CCRs from eurosids I. AtrCCR was used as the root. Recent whole genome duplications are marked with blue-filled ellipses (PDF 570 kb)

Online Resource 9 Catalytic residues for all detected proposed genuine CCRs in plant kingdom (XLSX 18 kb)

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Chao, N., Li, N., Qi, Q. et al. Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR. Planta 245, 61–75 (2017). https://doi.org/10.1007/s00425-016-2591-6

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