Isupov Michail N, Boyko Konstantin M, Sutter Jan-Moritz, James Paul, Sayer Christopher, Schmidt Marcel, Schönheit Peter, Nikolaeva Alena Yu, Stekhanova Tatiana N, Mardanov Andrey V, Ravin Nikolai V, Bezsudnova Ekaterina Yu, Popov Vladimir O, Littlechild Jennifer A
Henry Wellcome Building for Biocatalysis, Biosciences, University of Exeter, Exeter, United Kingdom.
Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Front Bioeng Biotechnol. 2019 Jan 24;7:7. doi: 10.3389/fbioe.2019.00007. eCollection 2019.
Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, , and . These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to ()-methylbenzylamine but no activity to ()-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the enzyme and 2.0 Å for the enzyme. Also the enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another () selective class IV transaminase from the fungus which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.
在不同嗜热古菌的基因组中已鉴定出两种新的嗜热支链氨基酸转氨酶，分别为[具体名称1]、[具体名称2]和[具体名称3]。根据其结构折叠，这些酶属于转氨酶的IV类。这些酶已被克隆并在[具体宿主]中过表达，并且对重组酶进行了生化和结构表征。两种酶均表现出高热稳定性，活性的最佳温度分别为80°C和85°C。在接触50%的有机溶剂乙醇、甲醇、二甲基亚砜和乙腈后，它们仍保留良好的活性。这些酶对（）-甲基苄胺活性较低，但对（）-甲基苄胺无活性。两种酶均已结晶，其结构以内部醛亚胺形式解析，[具体酶1]的分辨率为1.9 Å，[具体酶2]的分辨率为2.0 Å。此外，已确定[具体酶1]与氨基受体α-酮戊二酸形成的复合物以及[具体酶2]与抑制剂加巴喷丁形成的复合物的结构。这两种复合物有助于确定酶促反应过程中酶的构象，并增进了对其底物特异性的理解。已与先前研究的与加巴喷丁形成复合物的来自真菌[具体真菌名称]的另一种（）选择性IV类转氨酶进行了比较。这些酶之间细微的结构差异为它们不同的底物特异性提供了见解。
