Paris polyphylla

Yin, Yan, Gao, Linhui, Zhang, Xianan & Gao, Wei, 2018, A cytochrome P 450 monooxygenase responsible for the C- 22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway, Phytochemistry 156, pp. 116-123 : 118

2.2. Cloning of a Paris polyphylla homologue of the steroid C-22 hydroxylase gene

The entire cDNA sequence of the candidate gene c26719 was cloned and completely sequenced. The open reading frame of c26719 (GenBank: KX904822) is 1455 bp in length and encodes a protein of 484 amino acids with a molecular weight (MW) of 54,894.74 Da and a theoretical isoelectric point of 8.36. InterproScan analysis revealed that the protein encoded by c26719 belongs to the CYP450 superfamily, Eclass, group I with oxidoreductase activity. Analysis using the ScanProsite tool showed that the protein contains a cytochrome P450 cysteine heme-iron ligand signature (FSGGPRLCPG) at the position 422–431 aa ( Fig. S1 ). The neighbor-joining tree of C-22 hydroxylase showed that the amino acid sequence of c26719 is most closely related to an orthologue from V. californicum ( Fig. 3 ), a plant that also belongs to the Liliaceae and whose major secondary metabolites are steroid alkaloids. The BLAST results showed that the amino acid sequence of c26719 shared 82% identity with VcCYP90B27v1 (AJT59558.1), which prompted us to designate it as PpCYP90B27. Phylogenetic comparisons indicate that PpCYP90B27 is located in the cluster of the CYP90B subfamily, comprising the CYP90Bs that are responsible for the brassinosteroid biosynthesis pathway ( Fig. 3 ), but has no more than 55% homology to other members, with especially different amino acids in the cytochrome P450 cysteine heme-iron ligand signature composition ( Fig. S1 ). This suggests that PpCYP90B27 is involved in steroid C-22 hydroxylation, but the exact metabolic pathway it is specifically involved was uncertain.

Figures

Fig. 1. Biosynthetic pathway of spirostane-type saponins in plants. The solid- and dashed lines indicate single- and multiple catalytic steps, respectively. The enzyme shown in red was identified in this study. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Phylogenetic analysis of the amino acid sequences of steroid C-22 hydroxylases. This analysis involved 16 homologs from the following species: Os (Oryza sativa japonica group), Sl (Solanum lycopersicum), Pe (Populus euphratica), Gh (Gossypium hirsutum), Mt (Medicago truncatula), Cc (Cajanus cajan), Ep (Echinacea purpurea), At (Arabidopsis thaliana), Zm (Zea mays), Pp (Paris polyphylla), Vc (Veratrum californicum), St (Solanum tuberosum), Ar (Ajuga reptans), and Bm (Bombyx mori). The PpCYP90B29 from this study is highlighted by a red diamond shape. The evolutionary analyses were conducted in MEGA6 using the neighbor-joining method, and the percentage of replicate trees in which the associated taxa clustered together among the 1000 replicates in the bootstrap test are shown next to the branches. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)