Abstract
Thymidylate synthase (TS) catalyzes the synthesis of deoxythymidine monophosphate (dTMP), which is an essential precursor for DNA synthesis. The rationale underlying drug design is to identify compounds that differentially inhibit a viral or parasite enzyme vs. the host homologue. We studied the TS of the white spot syndrome virus (WSSV TS) and the corresponding TS from the host, the marine invertebrate shrimp Litopenaeus vannamei. TS is the only de novo source of dTMP and is essential for host and viral DNA replication. To establish proof of principle, we cloned a full-length TS cDNA from the white shrimp L. vannamei (shrimp TS) that corresponds to a deduced sequence of 289 amino acids and over-expressed it to study inhibition of both shrimp and viral TSs. Steady-state kinetic parameters for both TSs are similar, and dissociation (Kd) or half maximal inhibitory concentration constants (IC50) did not show differential inhibition between the folate analogues. Differences in their amino acid sequence are not reflected in theoretical molecular models of both TSs, since both appear to have identical active sites. These results suggest that the eukaryotic TS active site is very constrained into the functional residues involved in reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP).
Original language | English |
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Pages (from-to) | 406-413 |
Number of pages | 8 |
Journal | Comparative Biochemistry and Physiology - C Toxicology and Pharmacology |
Volume | 150 |
Issue number | 3 |
DOIs | |
State | Published - Sep 2009 |
Bibliographical note
Funding Information:This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) México D.F. grant 48991-Z to R. Sotelo-Mundo. A. Arvizu-Flores received a graduate scholarship from the CONACYT. Author A. Arvizu-Flores designed and performed experiments, analyzed data and wrote the paper; E. Aispuro-Hernandez and K. Garcia-Orozco designed and performed experiments, and reviewed the paper; A. Varela-Romero analyzed data and reviewed the paper; E. Valenzuela-Soto, A. Rojo-Dominguez and E. Velazquez-Contreras discussed and designed experiments, contributed techniques and reviewed the paper, F. Maley and G. Yepiz-Plascencia designed experiments, wrote and reviewed the paper, R. Sotelo-Mundo designed experiments, analyzed data, wrote and reviewed the paper. We thank Dr. Maria Islas-Osuna and Alonso Lopez-Zavala, M.Sc. for reviewing and suggesting improvements to the manuscript, and Monica Villegas, Salvador Carrasco and Monica Resendiz for technical support.
Keywords
- Antifolate
- Antifolate binding
- Differential inhibition
- Litopenaus vannamei
- Prawn
- Shrimp
- Thymidylate synthase
- White spot syndrome virus