Identification of novel thermostable taurine-pyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis
Identification of novel thermostable taurine-pyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis
复制标题
从热脱氮地芽孢杆菌中鉴定新型热稳定性牛磺酸-丙酮酸转氨酶，用于手性胺合成。
DOI：
10.1007/s00253-015-7129-5
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发表时间：
2016-04-01
影响因子：
5
通讯作者：
Wei, Dongzhi
中科院分区：
文献类型：
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作者：
Chen, Yujie;Yi, Dong;Wei, Dongzhi
ω-转氨酶（omega-TAs）是手性胺生物合成中最受欢迎的候选酶之一。确定尚未鉴定的ω-TA对于扩大其合成应用的潜力是重要的。牛磺酸-丙酮酸TA（TPTA，EC 2.6.1.77）是属于TA的III类的ω-TA。在这项研究中，我们克隆了一个新的热稳定性TPTA从嗜热土芽孢杆菌（TPTA（gth）），并在大肠杆菌中过表达。该酶在pH9.0和65 A ℃时活性最高，具有显著的热稳定性和对有机溶剂的耐受性。牛磺酸的K（M）和v（max）值分别为5.3 mM和0.28 μ mol s（-1）mg（-1）。底物耐受性的测定表明其对非天然底物的广泛供体和受体范围。值得注意的是，该酶显示出相对较好的活性，对酮糖，这表明其潜在的催化手性氨基醇的不对称合成。通过蛋白质序列比对、三维结构模拟和辅酶磷酸吡哆胺对接等方法，确定了TPTA（gth）的活性位点。TPTA（gth）的蛋白质序列和结构与3 N5 M亚家族的TA相似。发现其活性部位是其特殊的大口袋和基底隧道。此外，TPTA（gth）显示了一种独特的磺酸盐/α-羧酸盐识别机制，由Arg 163和Gln 160贡献。我们还确定了3 N5 M亚家族中TPTA的蛋白质序列指纹，其中涉及Arg 163和Gln 160以及413至419之间的7个额外残基，并且缺乏Phe/Tyr 22、Phe 85和Arg 409。
omega-Transaminases (omega-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified omega-TAs is important to broaden their potential for synthetic application. Taurine-pyruvate TA (TPTA, EC 2.6.1.77) is an omega-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTA(gth)) and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 9.0 and 65 A degrees C, with remarkable thermostability and tolerance toward organic solvents. Its K (M) and v (max) values for taurine were 5.3 mM and 0.28 mu mol s(-1) mg(-1), respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTA(gth) was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTA(gth) were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTA(gth) showed a unique mechanism of sulfonate/alpha-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.