The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction
The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction
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DOI：
10.1016/j.ijbiomac.2008.03.001
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发表时间：
2008-06-01
影响因子：
8.2
通讯作者：
Barile, Maria
中科院分区：
文献类型：
--
作者：
Brizio, Carmen;Brandsch, Roderich;Barile, Maria
大鼠线粒体黄素酶二甲基甘氨酸脱氢酶（Me（2）GlyDH）的前体在大肠杆菌中以C-末端6-His-标记的融合蛋白形式产生，通过一步亲和层析纯化并通过ESI-MS/MS鉴定。通过免疫学和荧光检测共价结合的黄素腺嘌呤二核苷酸（FAD）证明，纯化的前体主要是其脱辅基形式。这里描述的结果明确地证明：（i）FAD与Me的共价连接（2）GlyDH脱辅基酶可以在体外自催化地进行，而不需要第三反应物;（ii）通过线粒体加工肽酶去除线粒体前序列对于共价自身黄素化是不需要的。(c)2008 Elsevier B. V.保留所有权利。
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. (c) 2008 Elsevier B.V. All rights reserved.