Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-å crystal structure.
Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti-parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L. major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata. The L. major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity. The L. major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl beta-D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a bound Ca(2+) ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241. The structure is similar to the C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similarities, the catalytic specificities differ substantially. Relative values of k(cat) for the L. major enzyme with inosine, adenosine, guanosine, uridine, and cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C. fasciculata are 100, 15, 14, 510, and 36 for the same substrates. Iminoribitol analogues of the transition state are nanomolar inhibitors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.
Shi
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Albert Einstein College of Medicine, Department of Biochemistry, Bronx, New York 10461, USA.
Schramm
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Almo
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D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D017125:Genes, Protozoan; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D015800:Protozoan Proteins; D009699:N-Glycosyl Hydrolases