Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Structural Biology Research Center, Institute of Materials Structure Science, High Energy Accelerator Research Organization, KEK, 1-1 Oho, Tsukuba, Ibaraki, 305-0801, Japan.
Nat Commun. 2018 Jan 9;9(1):104. doi: 10.1038/s41467-017-02371-w.
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.
非血红素铁和α-酮戊二酸（αKG）加氧酶使用单一亚铁离子辅因子催化非常多样化的反应。研究这个多功能酶家族的主要挑战是了解它们的结构-功能关系。来自构巢曲霉的 AusE 和来自巴西青霉的 PrhA 是真菌生源异戊二烯途径中两种高度同源的 Fe(II)/αKG 加氧酶，它们使用 preustinoid A1 作为共同底物，催化不同的重排反应，分别形成 austinol 中的螺内酯和 paraherquonin 中的环庚二烯部分。本文报道了 AusE 和 PrhA 的比较结构研究，这导致了鉴定控制其反应性的三个关键活性位点残基。这些残基的结构导向诱变导致 AusE 和 PrhA 功能的成功转换，以及具有扩展催化谱的 PrhA 双突变体和三突变体的产生。多功能 Fe(II)/αKG 加氧酶的操作因此为未来生物催化剂的发展提供了一个极好的平台。
