title: Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR.
abstract: 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.
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors: Nan
authors: N
authors: Chao
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors: Ning
authors: N
authors: Li
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors: Qi
authors: Q
authors: Qi
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors: Shuang
authors: S
authors: Li
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors: Tong
authors: T
authors: Lv
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.|The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration, Beijing, 100083, China.
authors: Xiang-Ning
authors: XN
authors: Jiang
authors: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China. gaiying@bjfu.edu.cn.|The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration, Beijing, 100083, China. gaiying@bjfu.edu.cn.
authors: Ying
authors: Y
authors: Gai
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D032461:Chromosomes, Plant / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D057075:Enzyme Assays; D019143:Evolution, Molecular; D020440:Gene Duplication; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D062105:Molecular Docking Simulation; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D010802:Phylogeny; D032107:Populus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D000445:Aldehyde Oxidoreductases; C019904:cinnamoyl CoA reductase
keywords: Binding sites; Cinnamoyl-coenzyme A reductase; Gene family; Populus tomentosa; Site-directed mutations