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 language | English |
|---|---|
| Pages (from-to) | 69-76 |
| Number of pages | 8 |
| Journal | Journal of Biochemistry |
| Volume | 163 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2018 Jan 1 |
| Externally published | Yes |
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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 journal › Article
}
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 -