大鼠线粒体二甲基甘氨酸脱氢酶的纯化重组前体通过自催化反应结合黄素腺嘌呤二核苷酸（FAD）。

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction.

作者信息

Brizio Carmen, Brandsch Roderich, Douka Maria, Wait Robin, Barile Maria

机构信息

Dipartimento di Biochimica e Biologia Molecolare E. Quagliariello, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy.

出版信息

Int J Biol Macromol. 2008 Jun 1;42(5):455-62. doi: 10.1016/j.ijbiomac.2008.03.001. Epub 2008 Mar 13.

DOI:10.1016/j.ijbiomac.2008.03.001

PMID:18423846

Abstract

The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.

摘要

大鼠线粒体黄素酶二甲基甘氨酸脱氢酶（Me(2)GlyDH）的前体已在大肠杆菌中作为C末端带有6个组氨酸标签的融合蛋白产生，通过一步亲和层析纯化，并通过电喷雾串联质谱（ESI-MS/MS）鉴定。与溶解的大鼠肝线粒体共同孵育后，它被正确加工成成熟形式。通过对共价结合的黄素腺嘌呤二核苷酸（FAD）进行免疫和荧光检测表明，纯化的前体主要处于脱辅基形式。此处描述的结果明确证明：（i）FAD与Me(2)GlyDH脱辅酶的共价连接可以在无第三种反应物的情况下进行体外自催化；（ii）线粒体加工肽酶去除线粒体前导序列对于共价自黄素化不是必需的。