Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases, College of Life Sciences, Anhui Normal University, Wuhu, Anhui, 241000, China.
College of Biotechnology and Pharmaceutical Engineering, West Anhui University, Lu'an, Anhui, 237012, China.
World J Microbiol Biotechnol. 2024 Oct 19;40(11):357. doi: 10.1007/s11274-024-04169-7.
Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70 kDa. They are completely dependent on the coenzyme NADP and are inactive towards NAD. KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn and at pH 7.8 in the presence of Mg. Its optimal catalytic performance is achieved with both ions at 55 °C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn and Mg, respectively, and exhibits optimal catalytic performance at 45 °C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45 °C for 20 min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP to NAD by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies.
肺炎克雷伯菌和嗜肺军团菌是常见的革兰氏阴性菌，可引起肺部感染。肺炎克雷伯菌的多重耐药性对治疗构成了重大挑战。本研究专注于异柠檬酸脱氢酶（IDH），这是两种细菌氧化代谢途径中的关键酶。成功地过表达和纯化了 KpIDH 和 LpIDH，并对其生化特性进行了深入研究。研究表明，KpIDH 和 LpIDH 是同源二聚体酶，分子量约为 70 kDa。它们完全依赖辅酶 NADP，对 NAD 无活性。KpIDH 在存在 Mn 的情况下，在 pH 8.0 时表现出最高的催化活性，在存在 Mg 的情况下，在 pH 7.8 时表现出最高的催化活性。其最佳催化性能在 55°C 下，两种离子均达到最佳。LpIDH 在存在 Mn 和 Mg 的情况下，在 pH 7.8 时表现出最高的活性，在 45°C 时表现出最佳的催化性能。热失活研究表明，KpIDH 和 LpIDH 在 45°C 下暴露 20 分钟后，仍保留超过 80%的活性。此外，我们通过替换四个关键氨基酸残基成功地将 KpIDH 和 LpIDH 的辅酶特异性从 NADP 改变为 NAD。本研究全面表征了两种常见于医院环境中的多药耐药细菌 IDH，增强了我们对致病菌特性的理解，为开发新的治疗策略提供了参考。
