Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani

Abstract

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 k cat . 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 AMPbinding 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 α-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.

Computational Elucidation of Structural Basis for Ligand Binding withLeishmania donovaniAdenosine Kinase

“…Interestingly, all the six molecules form dataset O (namely, O1 to O6) and N1 showed prominent polar contact with Arg131 as conferred from our docking studies. In another report Datta et al have shown that mutation of Arg131 causes significant reduction in catalytic activity of Ld Adk [52]. Moreover they also concluded that Asp299 and Arg131 both are key catalytic residues in the protein; similar inference has been highlighted from our docking studies (Figure 4).…”

“…Moreover they also concluded that Asp299 and Arg131 both are key catalytic residues in the protein; similar inference has been highlighted from our docking studies (Figure 4). The diglycyl motif (formed by consecutive glycine residues: Gly61 and Gly62) present in the active site are responsible for maintaining conformation flexibility [52] and thus was thereason the rmsf data from modeled LdAdk showed high fluctuation as compared to other residues of the protein. In other reports from the same laboratory, they also established that Asp16 is also one of the crucial residues in the active site and is found in our study also where Asp16 is actively involved in forming H-bonds with the ligands.…”

The ADP‐dependent sugar kinase family: Kinetic and evolutionary aspects

“…In addition, mutation of this aspartic residue (D316) in mammalian adenosine kinase into either asparagine or glutamic acid, leads to a complete loss of activity (27). Similar results were obtained by mutation of the corresponding residue in the adenosine kinase from Leishmania donovani (D299) (28) and Homo sapiens (D300) (23). Also, the crystal structure of Toxoplasma gondii adenosine kinase shows that the side chain of the conserved D318 is wellpositioned for proton subtraction of the adenosine 5 0 -hydroxyl group (29).…”

“…Also, the crystal structure of Toxoplasma gondii adenosine kinase shows that the side chain of the conserved D318 is wellpositioned for proton subtraction of the adenosine 5 0 -hydroxyl group (29). On the basis of the crystallographic data and by comparison with the catalytic mechanism of E. coli ribokinase, D251 was also postulated as the catalytic base in the 2-keto-3-deoxygluconate kinase from Thermus termophilus (28). As a conserved feature, a contact has always been seen by X-ray crystallography between this conserved aspartic residue and the hydroxyl group that will receive the phosphate group in the phosphoryl acceptor molecule.…”

Adenosine Kinase ofTrypanosoma bruceiand Its Role in Susceptibility to Adenosine Antimetabolites

“…Since this is the case for most nucleoside antimetabolites, particular pharmacological importance is allocated to nucleoside kinases from parasites. Adenosine kinase has been dissected to the molecular level in T. gondii and Leishmania donovani (9,10,12,13,22,23,48), cloned from Babesia canis (5) and Cryptosporidium parvum (20), and characterized in cell extracts of Trypanosoma cruzi (28) and Eimeria spp. (36) but has not yet been described for African trypanosomes.…”