Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase

The Toxoplasma gondii nucleoside triphosphate hydrolase is the most active E-type ATPase yet identified, and was the first member of this new gene fam ily to be cloned (Bermudes D, Peck KR, Afifi-Afifi M, Beckers CJM, Joiner K A. J Biol Chem 1994;269:29252-29260. Previous work also identified two isof orms of the enzyme in the Virulent RH strain, and demonstrated that interna l fragments of the genes encoding these isoforms were found differentially in virulent Versus avirulent organisms (Asai T, Miura S, Sibley D, Okabayas hi H, Tsutomu T, J Biol Chem 1995;270:11391-11397). We now show that the NT Pase 1 isoform is expressed in avirulent strains, whereas virulent strains express both the NTPase 1 and NTPase 3 isoforms. The avirulent PLK strain l acks the gene for NTPase 3, explaining the absence of expression. Despite t he fact that NTPase 1 and NTPase 3 are 97% identical at the amino acid leve l, recombinant NTPase 1 is a true apyrase, whereas recombinant NTPase 3 cle aves predominantly nucleotide triphosphates. Furthermore, native and recomb inant NTPase 3 but neither native nor recombinant NTPase 1 bind to ATP-agar ose, further distinguishing the two isoforms. Using chimeras between the NT P1 and NTP3 genes, we show that a block of twelve residues at the C-terminu s dictates substrate specificity. These residues lie outside the regions co nserved among other E-ATPases, and therefore provide new insight into subst rate recognition by this class of enzymes. (C) 1998 Published by Elsevier S cience B.V. All rights reserved.