Helicobacter pylori Dihydroorotate Dehydrogenase: a Promising Target for Screening Potential Drug Candidates

Authors
Saba Ghaffari
Maryam Esmaeili
Fatemeh Ayoubnejad
Yeganeh Talebkhan
Marjan Mohammadi
Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
10.61882/ibj.5231
Abstract
Background: De novo pyrimidine biosynthesis is essential for the survival of all living organisms. DHODH catalyzes the fourth step in this pathway. Inhibition of DHODH induces pyrimidine depletion and effectively eradicates microorganisms like H. pylori, which lacks the pyrimidine salvage pathway. Herein, we expressed rHp-DHODH, characterized its enzymatic activity, kinetics, and stability, and subsequently evaluated its inhibition by HQNO.

Methods: The gene fragment encoding the rHp-DHODH protein was synthesized, subcloned, and expressed in soluble form. The recombinant protein was purified, its identity was confirmed, and its activity was measured using a colorimetric reduction assay. Kinetic parameters and the effects of pH, temperature, and incubation time on the enzymatic activity were investigated. The inhibitory effect of HQNO on rHp-DHODH was evaluated using the DCIP reduction assay. The MIC of HQNO against H. pylori was determined, and its bactericidal/bacteriostatic effect was assessed.

Results: Optimal soluble expression of rHp-DHODH was achieved in BL21(DE3)pLysS. The enzyme exhibited a specific activity of 5.7 U/mg. Maximum activity was observed at pH 8.0 in Tris-HCl buffer at 25 °C. The Km values were 39.75 µM for DHO and 5.37 µM for CoQ10, with a kcat of

3.82 s-¹. The IC50 of HQNO against the recombinant enzyme was determined as 1.75 μM. HQNO inhibited H. pylori growth with an MIC of 0.5-1.0 µg/mL, displaying concentration-dependent bacteriostatic to bactericidal effects.

Conclusions: The rHp-DHODH and its optimized enzymatic assay provide a reliable platform for screening candidate inhibitors, such as HQNO, advancing drug development efforts against H. pylori infection.
Keywords
Dihydroorotate dehydrogenase
Enzymes
Kinetics