Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation

Chisato Noguchi, Sho Miyazaki, Hiroshi Kawaide, Osamu Gotoh, Yuzo Yoshida, Yuri Aoyama

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

CYP701B1 of the moss, Physcomitrella patents, might be a unique cytochrome P450 having the ent-kaurene oxidase (KO) activity occurring in nonvascular plant. Phylogenetic analysis suggested that the gene encoding CYP701B1 was diverged from a common ancestral gene encoding KO of vascular plants. CYP701B1 expressed in Phichia yeast microsomes was purified and characterized. The purified CYP701B1 catalyzed the oxidation of ent-kaurene to entkaurenoic acid through three successive monooxygenations, and the rate-limiting step of this oxidation might be the initial step that forms ent-kaurenol. CYP701B1 was a typical ferric low-spin cytochrome P450 and was completely moved to high-spin state upon binding with entkaurene, and apparent Kd of ent-kaurene estimated by the spectral change caused by this spin-state shift was 2.5kM. The potent KO inhibitor uniconazole, an azole compound with molecular size similar to ent-kaurene, bound CYP701B1 with high affinity. However, ketoconazole, an azole compound whose molecular size is larger than ent-kaurene could not bind to CYP701B, though it binds strongly with CYP51, lanosterol 14-demethylase. The results indicated that the active site of CYP701B1 is fitted for the molecular size of ent-kaurene. The P450 monooxygenase adapted for ent-kaurene oxidation might appear in land plants before evolutionary divergence into vascular and nonvascular plants.

Original languageEnglish
Pages (from-to)69-76
Number of pages8
JournalJournal of Biochemistry
Volume163
Issue number1
DOIs
Publication statusPublished - 2018 Jan 1
Externally publishedYes

Keywords

  • CYP701B1
  • cytochrome P450
  • ent-kaurene oxidase
  • molecular evolution
  • nonvascular plant

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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