Gonorrhoea, caused by Neisseria gonorrhoeae, is a major global public health concern. Homoserine dehydrogenase (HSD), a key enzyme in the aspartate pathway, is a promising metabolic target against pathogenic infections. In this study, a monofunctional HSD from N. gonorrhoeae (NgHSD) was overexpressed in Escherichia coli and purified to >95% homogeneity for biochemical characterization. Unlike the classic dimeric structure, the purified recombinant NgHSD exists as a tetramer in solution. We determined the enzymatic activity of recombinant NgHSD for l-homoserine oxidation, which revealed that this enzyme was NAD⁺ dependent, with an approximate 479-fold (k_cat/K_m) preference for NAD⁺ over NADP⁺, and that optimal activity for l-homoserine oxidation occurred at pH 10.5 and 40 °C. At 800 mM, neither NaCl nor KCl increased the activity of NgHSD, in contrast to the behavior of several reported NAD⁺-independent homologs. Moreover, threonine did not markedly inhibit the oxidation activity of NgHSD. To gain insight into the cofactor specificity, site-directed mutagenesis was used to alter coenzyme specificity. The double mutant L45R/S46R, showing the highest affinity for NADP⁺, caused a shift in coenzyme preference from NAD⁺ to NADP⁺ by a factor of ~974, with a catalytic efficiency comparable with naturally occurring NAD⁺-independent homologs. Collectively, our results should allow the exploration of drugs targeting NgHSD to treat gonococcal infections and contribute to the prediction of the coenzyme specificity of novel HSDs.

由淋病奈瑟菌引起的淋病是全球主要的公共卫生问题。高丝氨酸脱氢酶 (HSD) 是天冬氨酸途径中的一种关键酶，是一种很有前景的抗病原体感染的代谢靶点。在本研究中，来自淋病奈瑟菌(NgHSD) 的单功能 HSD 在大肠杆菌中过表达并纯化至 > 95% 的同质性用于生化表征。与经典的二聚体结构不同，纯化的重组 NgHSD 在溶液中以四聚体的形式存在。我们测定了重组 NgHSD 对l-高丝氨酸氧化的酶活性，结果表明这种酶是 NAD ⁺依赖性的，大约 479 倍 ( k _cat /K _m ) 对 NAD ⁺的偏好优于 NADP ^{+ ，并且}l-高丝氨酸氧化的最佳活性发生在 pH 10.5 和 40 °C。在 800 mM 时，NaCl 和 KCl 都不会增加 NgHSD 的活性，这与一些报道的 NAD ⁺非依赖性同源物的行为相反。此外，苏氨酸对 NgHSD 的氧化活性没有明显抑制作用。为了深入了解辅酶特异性，使用定点诱变来改变辅酶特异性。^{双突变体 L45R/S46R，表现出对 NADP +}的最高亲和力，导致辅酶偏好从 NAD ⁺转变为 NADP ⁺约 974 倍，其催化效率可与天然存在的 NAD ⁺非依赖性同源物相媲美。总的来说，我们的结果应该允许探索针对 NgHSD 治疗淋球菌感染的药物，并有助于预测新型 HSD 的辅酶特异性。