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 journalArticle

1 Citation (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

Fingerprint

Kaurane Diterpenes
Bryophyta
Azoles
Gene encoding
Oxidation
Cytochrome P-450 Enzyme System
Blood Vessels
Bryopsida
Lanosterol
Embryophyta
Ketoconazole
Patents
Microsomes
Mixed Function Oxygenases
Yeast
Genes
ent-kaurene oxidase
Catalytic Domain
Yeasts
Acids

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation. / Noguchi, Chisato; Miyazaki, Sho; Kawaide, Hiroshi; Gotoh, Osamu; Yoshida, Yuzo; Aoyama, Yuri.

In: Journal of Biochemistry, Vol. 163, No. 1, 01.01.2018, p. 69-76.

Research output: Contribution to journalArticle

Noguchi, Chisato ; Miyazaki, Sho ; Kawaide, Hiroshi ; Gotoh, Osamu ; Yoshida, Yuzo ; Aoyama, Yuri. / Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation. In: Journal of Biochemistry. 2018 ; Vol. 163, No. 1. pp. 69-76.
@article{a07011339519440cb702f1f61ab9348e,
title = "Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation",
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.",
keywords = "CYP701B1, cytochrome P450, ent-kaurene oxidase, molecular evolution, nonvascular plant",
author = "Chisato Noguchi and Sho Miyazaki and Hiroshi Kawaide and Osamu Gotoh and Yuzo Yoshida and Yuri Aoyama",
year = "2018",
month = "1",
day = "1",
doi = "10.1093/jb/mvx063",
language = "English",
volume = "163",
pages = "69--76",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "1",

}

TY - JOUR

T1 - Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation

AU - Noguchi, Chisato

AU - Miyazaki, Sho

AU - Kawaide, Hiroshi

AU - Gotoh, Osamu

AU - Yoshida, Yuzo

AU - Aoyama, Yuri

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

KW - CYP701B1

KW - cytochrome P450

KW - ent-kaurene oxidase

KW - molecular evolution

KW - nonvascular plant

UR - http://www.scopus.com/inward/record.url?scp=85040658818&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85040658818&partnerID=8YFLogxK

U2 - 10.1093/jb/mvx063

DO - 10.1093/jb/mvx063

M3 - Article

C2 - 29036583

AN - SCOPUS:85040658818

VL - 163

SP - 69

EP - 76

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 1

ER -