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首页> 外文期刊>Applied Microbiology and Biotechnology >A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids
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A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids

机译：富营养的Ralstonia eutropha H16的丙酸酯CoA转移酶具有广泛的底物特异性，可催化各种羧酸的CoA硫酯形成

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In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoAtransferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64 kDa, respectively, and point to a quaternary structure of the native enzyme (α4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA–C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising-amongst others-the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16Δpct was generated. The growth and accumulation behaviour of thismutant on gluconate, gluconate plus 3,3′-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating other CoA-transferase(s) or CoA-synthetase(s), thereby compensating for the lacking Pct. The ability of R. eutropha H16 to substitute absent enzymes by isoenzymes has been already shown in different other studies in the past.
机译：在这项研究中，我们调查了富营养性拟南芥H16基因组中编码的丙酸CoA转移酶（Pct）同源物。已经将相应的基因克隆到载体pET-19b中，以产生在大肠杆菌BL21（DE3）中表达的组氨酸标签的酶。纯化后，高效液相色谱/质谱（HPLC / MS）分析显示该酶对羧酸具有广泛的底物特异性。使用丙酰基-CoA作为CoA供体形成乙酸的相应CoA-硫酯，并使用乙酰基-CoA作为CoA供体形成丙酸酯，丁酸酯，3-羟基丁酸酯，3-羟基丙酸酯，巴豆酸酯，丙烯酸酯，乳酸酯，琥珀酸酯和4-羟基丁酸酯。可以显示。根据底物特异性，可以将该酶分配到CoA转移酶的家族I中。通过凝胶过滤确定并通过SDS聚丙烯酰胺凝胶电泳检测的表观分子量分别为228和64kDa，并指向天然酶（α4）的四级结构。该酶在序列和结构上与丙酸梭菌的Pct具有相似性。它不包含典型的保守（S）ENG基序，但派生的基序序列带有谷氨酸342的EXG最有可能是催化残基。由于同型寡聚结构和与家族I CoA转移酶的IA-C子类的序列差异，提出了家族I的第四个子类，其中包括R. eutropha H16和C. propionicum的Pct。产生了无标记的精确缺失突变体富营养罗氏菌H16Δpct。研究了该突变体在葡萄糖酸盐，葡萄糖酸盐加3,3'-二硫代二丙酸（DTDP），乙酸盐和丙酸盐上的生长和积累行为，但未观察到表型。野生型和突变型都显示出与这些碳源相同的生长和储存行为。富营养芽孢杆菌H16可能会上调其他CoA转移酶或CoA合成酶，从而弥补缺乏的Pct。在过去的其他其他研究中，已经证明了富氧罗汉果H16可以被同工酶替代缺少的酶。

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