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.

Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family

“…The most striking homology between the predicted peptide sequences of OsCCRs and functional CCRs was found in regions covered by two highly conserved motifs. These were the NAD(P)-binding motif at the N-terminus, and the catalytic motif for CCR activity (Supplementary Figure 1 ) (Lacombe et al, 1997 ; Barakat et al, 2011 ; Chao et al, 2017 ). The former is a well-conserved motif for cofactor binding in the mammalian 3β-HSD/plant DFR protein superfamily (Baker et al, 1990 ; Baker and Blasco, 1992 ; Lacombe et al, 1997 ; Chao et al, 2017 ).…”

“…These were the NAD(P)-binding motif at the N-terminus, and the catalytic motif for CCR activity (Supplementary Figure 1 ) (Lacombe et al, 1997 ; Barakat et al, 2011 ; Chao et al, 2017 ). The former is a well-conserved motif for cofactor binding in the mammalian 3β-HSD/plant DFR protein superfamily (Baker et al, 1990 ; Baker and Blasco, 1992 ; Lacombe et al, 1997 ; Chao et al, 2017 ). The latter is a CCR signature motif (NWYCYGK), in which the NWYCY sequence may be crucial for CCR activity (Lacombe et al, 1997 ; Escamilla-Treviño et al, 2010 ; Barakat et al, 2011 ; Chao et al, 2017 ).…”

“…The former is a well-conserved motif for cofactor binding in the mammalian 3β-HSD/plant DFR protein superfamily (Baker et al, 1990 ; Baker and Blasco, 1992 ; Lacombe et al, 1997 ; Chao et al, 2017 ). The latter is a CCR signature motif (NWYCYGK), in which the NWYCY sequence may be crucial for CCR activity (Lacombe et al, 1997 ; Escamilla-Treviño et al, 2010 ; Barakat et al, 2011 ; Chao et al, 2017 ). Barakat et al ( 2011 ) applied maximum likelihood phylogenetic analysis to CCRs and CCR-like proteins from several land plant species, including A. thaliana , rice, P. trichocarpa , sorghum, grape ( Vitis vinifera ) and alfalfa ( Medicago truncatula ), and divided these proteins into three classes (Classes I–III).…”

“…Although OsCCR5 contained a fully conserved catalytic motif, it had no detectable activity toward the hydroxycinnamoyl-CoA substrate. This was likely caused by a polymorphism in the corresponding residue of H208, which is important in substrate binding as identified by a functional analysis of PtoCCRs ( P. tomentosa CCRs) (Supplementary Figure 1 ) (Chao et al, 2017 ). The polymorphism of H208 to A, R, M, V, K, L, M, and P residues are found in OsCCRs.…”

Benzaldehyde Synthases Are Encoded by Cinnamoyl-CoA Reductase Genes in Cucumber (Cucumis sativusL.)

“…Whether benzyol-CoA could be directly utilized as the precursor for benzaldehyde, and the last enzymatic step (benzaldehyde synthases, BS) remain to be addressed in plants. [19]. Given the similar chemical structure between C 6 -C 3 CoA ester (cinnamoyl-CoA in Fig.…”