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 ent-kaurenoic 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 ent-kaurene, and apparent Kd of ent-kaurene estimated by the spectral change caused by this spin-state shift was 2.5 μM. 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.

Multiple Metabolic Innovations and Losses Are Associated with Major Transitions in Land Plant Evolution

“…This supports previous reports that P. patens is missing KAO [7,46,47] and extends this finding to an additional moss species. The presence of KO in all land plants is consistent with observations that its metabolite product, the GA 12 intermediate KA, has been widely found in plants [48,49]. However, there has been no metabolomic evidence for the occurrence of recognized active forms of GA in bryophytes [47,50].…”

Insights into Heterologous Biosynthesis of Arteannuin B and Artemisinin in Physcomitrella patens

“…Endogenous P. patens oxidative enzymes fully convert amorpha-4,11-diene to artemisinic acid, since the alcohol and aldehyde intermediates were not detected in culture extracts. P. patens naturally produces high amounts of ent -kaurenoic acid from ent -kaurene, which is catalysed by an ent -kaurene oxidase (CYP701B1) through three successive oxidations [31,32]. CYP701B1 may therefore be a likely candidate for catalysing the conversion of amorpha-4,11-diene into artemisinic acid in P. patens.…”