Wang Fangfang, Lv Xiaoying, Guo Zhongyao, Wang Xianyi, Long Yaohang, Liu Hongmei
Biochemistry Teaching and Research Section, School of Basic Medical Sciences, Guizhou Medical University, Anshun 561113, China.
Engineering Research Center of Medical Biotechnology, School of Biology and Engineering, Guizhou Medical University, Anshun 561113, China.
Curr Issues Mol Biol. 2025 Aug 1;47(8):603. doi: 10.3390/cimb47080603.
Glutamate dehydrogenase (GDH) is ubiquitous in organisms and crucial for amino acid metabolism, energy production, and redox balance. The and genes encoding GDH were identified in AS19 and shown to be regulated by iron. However, their functions remain unclear. In this study, and were analyzed using bioinformatics tools, such as MEGA, Expasy, and SWISS-MODEL, expressed with a prokaryotic expression system, and the induction conditions were optimized to increase the yield of soluble proteins. Phylogenetic analysis revealed that GDH is evolutionarily conserved within the genus . GdhA and GudB were identified as hydrophobic proteins, not secreted or membrane proteins. Their structures were primarily composed of irregular coils and α-helices. SWISS-MODEL predicts GdhA to be an NADP-specific GDH, whereas GudB is an NAD-specific GDH. SDS-PAGE analysis showed that GdhA was expressed as a soluble protein after induction with 0.2 mmol/L IPTG at 24 °C for 16 h. GudB was expressed as a soluble protein after induction with 0.1 mmol/L IPTG at 16 °C for 12 h. The proteins were confirmed by Western blot and mass spectrometry. The enzyme activity of recombinant GdhA was 62.7 U/mg with NADPH as the coenzyme. This study provides a foundation for uncovering the functions of two GDHs of AS19.
谷氨酸脱氢酶（GDH）在生物体中广泛存在，对氨基酸代谢、能量产生和氧化还原平衡至关重要。在嗜盐放线菌AS19中鉴定出了编码GDH的gdhA和gudB基因，并表明它们受铁的调控。然而，它们的功能仍不清楚。在本研究中，使用MEGA、Expasy和SWISS-MODEL等生物信息学工具对gdhA和gudB进行了分析，用原核表达系统进行表达，并优化诱导条件以提高可溶性蛋白的产量。系统发育分析表明，GDH在嗜盐放线菌属内具有进化保守性。GdhA和GudB被鉴定为疏水蛋白，不是分泌蛋白或膜蛋白。它们的结构主要由不规则卷曲和α螺旋组成。SWISS-MODEL预测GdhA是一种NADP特异性GDH，而GudB是一种NAD特异性GDH。SDS-PAGE分析表明，GdhA在24℃用0.2 mmol/L IPTG诱导16 h后表达为可溶性蛋白。GudB在16℃用0.1 mmol/L IPTG诱导12 h后表达为可溶性蛋白。通过蛋白质免疫印迹和质谱对蛋白质进行了确认。以NADPH为辅酶时，重组GdhA的酶活性为62.7 U/mg。本研究为揭示嗜盐放线菌AS19的两种GDH的功能奠定了基础。
