Hirato Yuki, Tokuhisa Mayumi, Tanigawa Minoru, Ashida Hiroyuki, Tanaka Hiroyuki, Nishimura Katsushi
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda-Ku, Tokyo, 101-8308, Japan.
Department of Molecular and Functional Genomics, Interdisciplinary Center for Science Research, Shimane University, Nishikawatsu 1060, Matsue, Shimane, 690-8504, Japan.
Phytochemistry. 2017 Mar;135:18-23. doi: 10.1016/j.phytochem.2016.12.012. Epub 2016 Dec 27.
d-Threonine aldolase (DTA) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent interconversion of d-threonine and glycine plus acetaldehyde. The enzyme is a powerful tool for the stereospecific synthesis of various β-hydroxy amino acids in synthetic organic chemistry. In this study, DTA from the green alga Chlamydomonas reinhardtii was discovered and characterized, representing the first report to describe the existence of eukaryotic DTA. DTA was overexpressed in recombinant Escherichia coli BL21 (DE3) cells; the specific activity of the enzyme in the cell-free extract was 0.8 U/mg. The recombinant enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE-Sepharose, and Mono Q column chromatographies (purified enzyme 7.0 U/mg). For the cleavage reaction, the optimal temperature and pH were 70 °C and pH 8.4, respectively. The enzyme demonstrated 90% of residual activity at 50 °C for 1 h. The enzyme catalyzed the synthesis of d- and d-allo threonine from a mixture of glycine and acetaldehyde (the diastereomer excess of d-threonine was 18%). DTA was activated by several divalent metal ions, including manganese, and was inhibited by PLP enzyme inhibitors and metalloenzyme inhibitors.
d-苏氨酸醛缩酶（DTA）催化依赖于磷酸吡哆醛（PLP）的d-苏氨酸与甘氨酸及乙醛之间的相互转化。在合成有机化学中，该酶是立体特异性合成各种β-羟基氨基酸的有力工具。在本研究中，发现并表征了来自绿藻莱茵衣藻的DTA，这是描述真核生物DTA存在的首篇报道。DTA在重组大肠杆菌BL21（DE3）细胞中过表达；无细胞提取物中该酶的比活性为0.8 U/mg。通过硫酸铵分级分离、DEAE-琼脂糖和Mono Q柱色谱将重组酶纯化至同质（纯化酶7.0 U/mg）。对于裂解反应，最佳温度和pH分别为70℃和pH 8.4。该酶在50℃下保温1小时后仍具有90%的残余活性。该酶催化由甘氨酸和乙醛混合物合成d-苏氨酸和d-别苏氨酸（d-苏氨酸的非对映体过量为18%）。DTA被包括锰在内的几种二价金属离子激活，并被PLP酶抑制剂和金属酶抑制剂抑制。
