中国药科大学学报2026，Vol.57Issue(2)：240-245,6.DOI:10.11665/j.issn.1000-5048.2025082602
小分子化合物Lyb24与二氢乳清酸脱氢酶PyrD的相互作用研究
Study on the interaction between small molecule Lyb24 and dihydroorotate dehydrogenase PyrD
摘要
Abstract
This study aimed to explore the interaction between the small molecule Lyb24 and PyrD,a key enzyme in the pyrimidine biosynthesis pathway of Klebsiella pneumoniae(KP),and the effect of Lyb24 on the catalytic activity of PyrD,thus to provide a theoretical basis for the development of novel antimicrobial agents.The pET-30a(+)-PyrD recombinant plasmid was constructed using Nde I/Xba I double digestion technology and was transformed into Escherichia coli BL21(DE3)competent cells using the heat-shock method.The recombinant protein was induced at 16℃with 0.3 mmol/L isopropyl β-D-thiogalactopyranoside(IPTG).The r ecombinant PyrD protein was purified using nickel-nitrilotriacetic acid(Ni-NTA)affinity chromatography to obtain a high-purity product.Surface plasmon resonance(SPR)experiments were conducted to detect the direct interaction between Lyb24 and PyrD protein,and a DCIP-based colorimetric assay was used to evaluate the effect of Lyb24 on the catalytic activity of PyrD.The pET-30a(+)-PyrD plasmid was successfully constructed,and the recombinant PyrD protein with a molecular weight of approximately 36 kD was expressed and purified to a concentration of 5.58 mg/mL.Lyb24 exhibited high-affinity direct binding to PyrD(KD=8.83×10-5 mol/L)and exerted an uncompetitive inhibition effect on the catalytic activity of PyrD.This study demonstrates that Lyb24,a small-molecule compound,directly binds to PyrD and inhibits its enzymatic activity,providing crucial experimental evidence for developing PyrD-targeted antibacterial agents with value of clinical translation.关键词
肺炎克雷伯菌/抑制剂/Lyb24/PyrD/机制研究Key words
Klebsiella pneumoniae/inhibitor/Lyb24/PyrD/mechanism study分类
医药卫生引用本文复制引用
孙嘉荣,王淑燕,黄维,鲁超..小分子化合物Lyb24与二氢乳清酸脱氢酶PyrD的相互作用研究[J].中国药科大学学报,2026,57(2):240-245,6.基金项目
深圳市呼吸系统疾病临床医学研究中心课题(LCYSSQ20220823091203007) This study was supported by Shenzhen Clinical Research Center for Respiratory Disease(LCYSSQ20220823091203007) （LCYSSQ20220823091203007）
