Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of L-aspartate-4-semialdehyde to L5 homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes because it is known for its application in the development of pesticides and is being utilized in the large scale production of L-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for L-homoserine oxidation and found that BsHSD exclusively prefers NADP⁺ to NAD⁺ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. In kinetic analysis, K_m values for L-homoserine and NADP⁺ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the V_max values were 2.72 ± 0.06 μmol min^-1 mg^-1 and 2.79 ± 0.11 μmol min^-1 mg^-1, respectively. The apparent molecular mass determined with the size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from the other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. The thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was determined to be 54.8 °C. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.

中文翻译：

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枯草芽孢杆菌高丝氨酸脱氢酶的分子和酶学特征。

高丝氨酸脱氢酶 (HSD) 在赖氨酸、甲硫氨酸、苏氨酸和异亮氨酸生物合成的天冬氨酸途径中催化 L-天冬氨酸-4-半醛可逆转化为 L5 高丝氨酸。HSD 在医学和工业用途上引起了极大的关注，因为它以其在杀虫剂开发中的应用而闻名，并被用于 L-赖氨酸的大规模生产。在这项研究中，来自枯草芽孢杆菌(BsHSD) 的 HSD 在大肠杆菌中过表达，并纯化至同质性以用于生化表征。我们检查了 BsHSD 对 L-高丝氨酸氧化的酶活性，发现 BsHSD 只偏好 NADP ⁺而不是 NAD ⁺并且其活性在 pH 9.0 和存在 0.4 M NaCl 时最大。在动力学分析中，发现L-高丝氨酸和 NADP ^+的K _m值分别为 35.08 ± 2.91 mM 和 0.39 ± 0.05 mM，V_最大值分别为 2.72 ± 0.06 μmol min ^-1 mg ^-1和 2.79 ± 0.11 μmol min ^-1 mg ^-1， 分别。用尺寸排阻色谱测定的表观分子量表明 BsHSD 形成四聚体，这与之前报道的来自其他生物体的二聚体 HSD 形成对比。这种新型寡聚组装可归因于 BsHSD 的额外 C 端 ACT 结构域。利用圆二色谱法监测热变性，测定其熔点，经测定为54.8℃。本研究揭示的 BsHSD 的分子和生化特征可能为未来氨基酸代谢研究及其工业和医学应用奠定基础。