Functional identity of the active sites of crustacean and viral thymidylate synthases.
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 (K(d)) or half maximal inhibitory concentration constants (IC(50)) 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).
Arvizu-Flores
Aldo A
AA
Centro de Investigación en Alimentación y Desarrollo (CIAD), Hermosillo, Sonora, Mexico.
Aispuro-Hernandez
Emmanuel
E
Garcia-Orozco
Karina D
KD
Varela-Romero
Alejandro
A
Valenzuela-Soto
Elisa
E
Velazquez-Contreras
Enrique F
EF
Rojo-Domínguez
Arturo
A
Yepiz-Plascencia
Gloria
G
Maley
Frank
F
Sotelo-Mundo
Rogerio R
RR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology; D054833:Isoindoles / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000201:enzymology*; D010802:Phylogeny; D011799:Quinazolines / Q000494:pharmacology; D016415:Sequence Alignment; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D046848:White spot syndrome virus 1 / Q000201:enzymology*
D003856:Deoxyuracil Nucleotides; D005493:Folic Acid Antagonists; D054833:Isoindoles; D011799:Quinazolines; C079601:1843U89; C031662:CB 3717; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase