The Oxidation Cascade of a Rare Multifunctional P450 Enzyme Involved in Asperterpenoid A Biosynthesis.

Hui-Yun Huang,Jia-Hua Huang,Hao Gao,Zhi-Gang She,Yong-Heng Wang,Guo-Dong Chen,Yong-Jun Lu,Xin-Sheng Yao,Dan Hu

doi:10.3389/fchem.2021.785431

Abstract

The cytochrome P450 enzymes (P450s or CYPs) are heme-containing enzymes which catalyze a wide range of oxidation reactions in nature. In our previous study, a rare multifunctional P450 AstB was found, which can dually oxidize two methyl groups (C-19 and C-21) of preasperterpenoid A to asperterpenoid A with 3-carboxyl and 11-hydroxymethyl groups. However, the oxidation order of C-19 and C-21 catalyzed by AstB is unclear. In order to reveal this oxidation order, probable pathways catalyzed by AstB were proposed, and the oxidation order of C-19 and C-21 was obtained by quantum chemistry calculations. The potential intermediates (three new asperterpenoids D–F, 1–3) were obtained through the chemical investigation on the extract of the transformant strain and chemical conversions, which were used as the standards to detect their existences in the extract of the transformant strain with HPLC-MS. Combined with the quantum chemistry calculation and the HPLC-MS analysis, the catalyzed order of AstB in asperterpenoid A biosynthesis was revealed. Furthermore, the mPTPB inhibition of obtained asperterpenoids was evaluated, and the results showed that 3-carboxyl and the oxidation station of C-21 would be the key factors for mPTPB inhibition of asperterpenoids.

Highlights

Cytochrome P450 enzymes (P450s or CYPs) are a kind of enzyme catalyzing a wide range of oxidation reactions at the specific site of molecules, which play an important role in the metabolism of organisms (Sheng et al, 2009) and the biosynthesis of natural products with potent bioactivity (Jiang et al, 2021; Lin et al, 2019)
In our search of bioactive compounds from fungi through genome mining (Huang et al, 2019; Zhang et al, 2020), a rare oxidation multifunctional P450 AstB was found, which was solely responsible for the transformation of preasperterpenoid A to asperterpenoid A via C-19 and C-21 oxidations of preasperterpenoid A (Figure 1)
Because the oxidation orders of 3- and 11-methyls to hydroxymethyls and 3-hydroxymethyl to 3-carboxyl are unclear, there would be four potential intermediates, leading to three possible oxidation routes (A, B, and C) for the generation of asperterpenoid A from preasperterpenoid A catalyzed by AstB (Figure 2)

Summary

INTRODUCTION

Cytochrome P450 enzymes (P450s or CYPs) are a kind of enzyme catalyzing a wide range of oxidation reactions at the specific site of molecules, which play an important role in the metabolism of organisms (Sheng et al, 2009) and the biosynthesis of natural products with potent bioactivity (Jiang et al, 2021; Lin et al, 2019). In our search of bioactive compounds from fungi through genome mining (Huang et al, 2019; Zhang et al, 2020), a rare oxidation multifunctional P450 AstB was found, which was solely responsible for the transformation of preasperterpenoid A to asperterpenoid A (the molecule with 3-carboxyl and 11hydroxymethyl groups) via C-19 and C-21 oxidations of preasperterpenoid A (Figure 1). The oxidation order of C-19 and C-21 catalyzed by AstB remained obscure because no intermediate in the generation of asperterpenoid A was obtained from the heterologously expressed astBCharboring transformant strain (Aspergillus oryzae) with the previous fermentation condition (Huang et al, 2019). The mPTPB inhibition of obtained asperterpenoids was evaluated

MATERIALS AND METHODS

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT