A cytochrome P 450 monooxygenase responsible for the C- 22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway Author Yin, Yan School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China & School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, PR China Author Gao, Linhui School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China & State Key Laboratory of Breeding Base Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, Author Zhang, Xianan School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China xnzhang@ccmu.edu.cn Author Gao, Wei School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China text Phytochemistry 2018 2018-12-31 156 116 123 http://dx.doi.org/10.1016/j.phytochem.2018.09.005 journal article 10.1016/j.phytochem.2018.09.005 1873-3700 10484444 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.