Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-08 DOI:10.1016/bs.mie.2024.05.006
Daan Ren, Yu-Hsuan Lee, Hung-Wen Liu
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引用次数: 0

Abstract

Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.

草嗪霉素生物合成过程中的非血红素铁依赖性单氧化酶 OzmD 的表达、纯化和特征。
Oxazinomycin 是一种 C 核苷天然产物,其特征是 1,3-oxazine 环通过 C-C 糖苷键与核糖相连。1,3-oxazine 环的形成取决于 OzmD 的活性,OzmD 是一种单核非血红素铁依赖性酶,属于含有未知功能域(DUF)4243 的酶家族。OzmD 催化吡啶衍生物不寻常的氧化环重排,在恶嗪霉素生物合成的最后阶段释放出氰化物作为副产品。OzmD 底物对氧的内在敏感性以及催化作用对氧的依赖性给体外酶学检测带来了巨大挑战。本章介绍了表征 OzmD 的详细步骤,包括蛋白质制备、活性测定和反应副产物鉴定。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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