Giardia intestinalis deoxyadenosine kinase has a unique tetrameric structure that enables high substrate affinity and makes the parasite sensitive to deoxyadenosine analogues

Abstract

Giardia intestinalis is a protozoan parasite causing giardiasis, a severe, sometimes even life-threatening, diarrheal disease. Giardia is one of only a few known organisms that lack de novo synthesis of DNA building blocks, and the parasite is therefore completely dependent on salvaging deoxyribonucleosides from the host. The deoxyribonucleoside kinases (dNKs) needed for this salvage are generally divided into two structurally distinct families, thymidine kinase 1 (TK1)-like dNKs and non-TK1-like dNKs. We have characterized the G. intestinalis deoxyadenosine kinase and found that it, in contrast to previously studied non-TK1-like dNKs, has a tetrameric structure. Deoxyadenosine was the best natural substrate of the enzyme (KM=1.12 M; Vmax=10.3 mol{middle dot}min-1{middle dot}mg-1), whereas the affinities for deoxyguanosine, deoxyinosine and deoxycytidine were 400-2000 times lower. Deoxyadenosine analogues halogenated at the 2- and/or 2 s-positions were also potent substrates, with comparable EC50 values as the main drug used today, metronidazole, but with the advantage of being usable on metronidazole-resistant parasites. Cryo-EM and 2.1 [A] X-ray structures of the enzyme in complex with the product dAMP (and dADP) showed that the tetramer is kept together by extended N- and C-termini that reach across from one canonical dimer to the next in a novel dimer-dimer interaction. Removal of the two termini resulted in lost ability to form tetramers and a 100-fold decreased deoxyribonucleoside substrate affinity. This is the first example of a non-TK1-like dNK that has a higher substrate affinity as the result of a higher oligomeric state. The development of high substrate affinity could be an evolutionary key factor behind the ability of the parasite to survive solely on deoxyribonucleoside salvage. Authors summaryThe human pathogen Giardia intestinalis is one of only a few organisms that lack ribonucleotide reductase and is therefore completely dependent on salvaging deoxyribonucleosides from the host for the supply of DNA building blocks. We have characterized one of the G. intestinalis salvage enzymes, which was named deoxyadenosine kinase based on its substrate specificity. The enzyme also phosphorylated many deoxyadenosine analogues that were equally efficient in preventing parasite growth as the most used drug today, metronidazole, and also usable against metronidazole-resistant parasites. Structural analysis of the enzyme with cryo-EM and X-ray crystallography showed that the enzyme was unique in its family of deoxyribonucleoside kinases by forming a tetramer and mutational analysis showed that tetramerization is a prerequisite for the high substrate affinity of the enzyme. The ability to gain substrate affinity by increasing the number of enzyme subunits could potentially represent an evolutionary pathway that has assisted the parasite to become able to survive entirely on salvage synthesis of DNA building blocks.