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Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif.

ABSTRACT: The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5' RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.

SUBMITTER: Sinha KM

PROVIDER: S-EPMC1220203 | biostudies-other | 1999 May

REPOSITORIES: biostudies-other

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Project description:Leishmania donovani adenosine kinase (LdAdK) plays a pivotal role in scavenging of purines from the host. Exploiting interspecies homology and structural co-ordinates of the enzyme from other sources, we generated a model of LdAdK that led us to target several amino acid residues (namely Gly-62, Arg-69, Arg-131 and Asp-299). Replacement of Gly-62 with aspartate caused a drastic reduction in catalytic activity, with decreased affinity for either substrate. Asp-299 was found to be catalytically indispensable. Mutation of either Arg-131 or Arg-69 caused a significant reduction in kcat. R69A (Arg-69-->Ala) and R131A mutants exhibited unaltered K(m) for either substrate, whereas ATP K(m) for R69K increased 6-fold. Importance of both of the arginine residues was reaffirmed by the R69K/R131A double mutant, which exhibited approx. 0.5% residual activity with a large increase in ATP K(m). Phenylglyoxal, which inhibits the wild-type enzyme, also inactivated the arginine mutants to different extents. Adenosine protected both of the Arg-69 mutants, but not the R131A variant, from inactivation. Binding experiments revealed that the AMP-binding property of R69K or R69A and D299A mutants remained largely unaltered, but R131A and R69K/R131A mutants lost their AMP binding ability significantly. The G62D mutant did not bind AMP at all. Free energy calculations indicated that Arg-69 and Arg-131 are functionally independent. Thus, apart from the mandatory requirement of flexibility around the diglycyl (Gly-61-Gly-62) motif, our results identified Asp-299 and Arg-131 as key catalytic residues, with the former functioning as the proton abstractor from the 5'-OH of adenosine, while the latter acts as a bidentate electrophile to stabilize the negative charge on the leaving group during the phosphate transfer. Moreover, the positive charge distribution of Arg-69 probably helps in maintaining the flexibility of the alpha-3 helix needed for proper domain movement. These findings provide the first comprehensive biochemical evidence implicating the mechanistic roles of the functionally important residues of this chemotherapeutically exploitable enzyme.

Project description:Despite designating catalytic roles of Asp299 and Arg131 during the transfer of gamma-phosphate from ATP to Ado (adenosine) [R. Datta, Das, Sen, Chakraborty, Adak, Mandal and A. K. Datta (2005) Biochem. J. 387, 591-600], the mechanisms that determine binding of substrate and cause product inhibition of adenosine kinase from Leishmania donovani remained unclear. In the present study, employing homology-model-guided site-specific protein mutagenesis, we show that Asp16 is indispensable, since its replacement with either valine or arginine resulted in a >200-fold increase in K(m) (Ado) with a 1000-fold decrease in k(cat)/K(m), implying its critical importance in Ado binding. Even glutamate replacement was not tolerated, indicating the essentiality of Asp16 in the maintenance of steric complementarity of the binding pocket. Use of 2'or 3'-deoxygenated Ado as substrates indicated that, although both the hydroxy groups play important roles in the formation of the enzyme-Ado complex, the binding energy (DeltaDeltaG(B)) contribution of the former was greater than the latter, suggesting possible formation of a bidentate hydrogen bond between Asp16 and the adenosyl ribose. Interestingly, AMP-inhibition and AMP-binding studies revealed that, unlike the R131A mutant, which showed abrogated AMP-binding and insensitivity towards AMP inhibition despite its unaltered K(m) (Ado), all the Asp16 mutants bound AMP efficiently and displayed AMP-sensitive catalytic activity, suggesting disparate mechanisms of binding of Ado and AMP. Molecular docking revealed that, although both Ado and AMP apparently occupied the same binding pocket, Ado binds in a manner that is subtly different from AMP binding, which relies heavily on hydrogen-bonding with Arg131 and thus creates an appropriate environment for competition with Ado. Hence, besides its role in catalysis, an additional novel function of the Arg131 residue as an effector of product-mediated enzyme regulation is proposed.

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Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif.

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