Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate β-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD⁺ over NADP⁺, in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD⁺-dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius.

高丝氨酸脱氢酶（HSD），通过对编码坎基因，是在天门冬氨酸途径，其可逆地催化转化的关键酶升天冬氨酸β半醛到升高丝氨酸（升-Hse），即使用NAD（H）或NADP(H) 作为辅酶。在这项工作中，我们提出了共生多核杆菌必需亚种的 HSD 的第一个特征。必需的(PnHSD) 在大肠杆菌中产生。序列分析表明，PnHSD 是一个含有 ACT 结构域的单功能 HSD，具有 436 个羊酸残基。SDS-PAGE 和蛋白质印迹证明 PnHSD 可以在大肠杆菌中过表达通过使用 SUMO 融合技术将 BL21(DE3) 细胞作为可溶形式。它可以被纯化到明显的同质性以进行生化表征。尺寸排阻色谱显示纯化的 PnHSD 具有约 160 kDa 的天然分子质量，表明其为同四聚体结构。在这项工作中研究了 PnHSD 的氧化活性。动力学分析表明，PnHSD显示的高达1460倍偏好NAD ⁺在NADP ⁺，而相比之下，其同系物。纯化的 PnHSD 在 35 °C 和 pH 11 时表现出最大活性。与其 NAD ⁺相似依赖同系物，既没有观察到 NaCl 和 KCl 的激活，也没有观察到 L-Thr 对 PnHSD 酶活性的抑制。这些结果将有助于更好地了解 HSD 家族的辅酶特异性和P. necessarius的天冬氨酸途径。