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首页> 外文期刊>Applied Microbiology >Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16
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Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

机译：Ralstonia eutropha H16的两种半胱氨酸双加氧酶的底物和辅因子范围差异

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Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.
机译：半胱氨酸双加氧酶（Cdos）催化半胱氨酸向半胱氨酸亚磺酸（CSA）的硫氧化反应，由于其在几种疾病中的作用，已经在真核生物中进行了广泛研究。相反，仅研究了少数这种类型的原核酶。在富营养尔氏菌H16中，先前已鉴定出两个Cdo同源物（CdoA和CdoB）。体内研究表明，表达CdoA的大肠杆菌细胞可将3-巯基丙酸酯（3MP）转化为3-磺基丙酸酯（3SP），而在表达CdoB的细胞中未检测到3SP。这项研究的目的是证实这些发现，并通过进行体外表征来详细研究这两种酶。蛋白质通过固定的金属螯合亲和层析（IMAC）异源表达并纯化至表观同质性。随后的酶活性分析显示，在底物范围和对过渡金属辅因子的特异性方面存在显着差异，例如，CdoA催化3MP的硫氧化作用比半胱氨酸的硫氧化作用高3倍，而CdoB仅能转化半胱氨酸。此外，可以证明富营养杜鹃H16的Cdos活性对活性中心金属辅因子的依赖性。通过进一步转化降解产物3MP，证实了CdoA对于硫化合物3,3'-硫代二丙酸（TDP）和3,3'-二硫代二丙酸（DTDP）代谢的重要性。由于3MP还可作为富营养罗非鱼H16中聚硫酯（PTE）合成的前体，因此删除cdoA可能会增加PTE的合成。

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