Study on the interaction between small molecule Lyb24 and dihydroorotate dehydrogenase PyrD
10.11665/j.issn.1000-5048.2025082602
- VernacularTitle:小分子化合物Lyb24与二氢乳清酸脱氢酶PyrD的相互作用研究
- Author:
Jiarong SUN
1
;
Shuyan WANG
;
Wei HUANG
;
Chao LU
Author Information
1. 安徽理工大学第一附属医院, 淮南232007;深圳市人民医院检验科, 深圳518020;深圳市呼吸系统疾病临床医学研究中心, 深圳市呼吸疾病研究所, 深圳市人民医院, 深圳518020;安徽理工大学第一附属医院药物临床试验研究中心, 淮南232007
- Publication Type:Journal Article 期刊文章
- Keywords:
Klebsiella pneumoniae;
inhibitor;
Lyb24;
PyrD;
mechanism study
- From:
Journal of China Pharmaceutical University
2026;57(2):240-245
- CountryChina
- Language:Chinese
-
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 recombinant 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.
