Structure function and engineering of multifunctional non-heme iron dependent oxygenases in fungal meroterpenoid biosynthesis

Yu Nakashima,Miki Senda,Takahiro Mori,Ikuro Abe,Takayoshi Awakawa,Hitomi Nakamura,Toshiya Senda,Shotaro Hoshino

doi:10.1038/s41467-017-02371-w

Abstract

Non-heme iron and α-ketoglutarate (αKG) oxygenases catalyze remarkably diverse reactions using a single ferrous ion cofactor. A major challenge in studying this versatile family of enzymes is to understand their structure–function relationship. AusE from Aspergillus nidulans and PrhA from Penicillium brasilianum are two highly homologous Fe(II)/αKG oxygenases in fungal meroterpenoid biosynthetic pathways that use preaustinoid A1 as a common substrate to catalyze divergent rearrangement reactions to form the spiro-lactone in austinol and cycloheptadiene moiety in paraherquonin, respectively. Herein, we report the comparative structural study of AusE and PrhA, which led to the identification of three key active site residues that control their reactivity. Structure-guided mutagenesis of these residues results in successful interconversion of AusE and PrhA functions as well as generation of the PrhA double and triple mutants with expanded catalytic repertoire. Manipulation of the multifunctional Fe(II)/αKG oxygenases thus provides an excellent platform for the future development of biocatalysts.

Highlights

Non-heme iron and α-ketoglutarate oxygenases catalyze remarkably diverse reactions using a single ferrous ion cofactor
Non-heme iron and α-ketoglutarate-dependent dioxygenases are widely distributed in nature, and play a major role in diversifying the molecular scaffold
Many of the Fe(II)/αKGdependent oxygenases found in fungal meroterpenoid biosynthesis are multifunctional and a single enzyme catalyzes a variety of chemistry ranging from simple hydroxylations[12, 13] to remarkable skeletal rearrangements[13,14,15,16,17]

Summary

Introduction

Non-heme iron and α-ketoglutarate (αKG) oxygenases catalyze remarkably diverse reactions using a single ferrous ion cofactor. AusE from Aspergillus nidulans and PrhA from Penicillium brasilianum are two highly homologous Fe(II)/αKG oxygenases in fungal meroterpenoid biosynthetic pathways that use preaustinoid A1 as a common substrate to catalyze divergent rearrangement reactions to form the spiro-lactone in austinol and cycloheptadiene moiety in paraherquonin, respectively. Non-heme iron and α-ketoglutarate (αKG; called 2oxoglutarate)-dependent dioxygenases are widely distributed in nature, and play a major role in diversifying the molecular scaffold This superfamily of enzymes employs αKG as a co-substrate and Fe(II) as a cofactor to couple substrate oxidation to concomitant decarboxylation of αKG to form succinate and CO2. PrhA first desaturates at C5–C6 to form berkeleyone B (11), followed by rearrangement of the A/B-ring to form the cycloheptadiene moiety in berkeleydione (12) (Fig. 1 and Supplementary Fig. 1c) Such functional difference between AusE and PrhA is not apparent from simple comparison of their primary sequences. Structural study and engineering of Fe(II)/αKG oxygenases that catalyze complex multistep oxidations serve as an important milestone in future efforts to understand and control the reactivity of these enzymes

Methods

Results

Conclusion