Enhancing nicotinamide N-methyltransferase bisubstrate inhibitor activity through 7-deazaadenosine and linker modifications.

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3-12 (IC50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors.

Discovery and characterization of the tubercidin biosynthetic pathway from Streptomyces tubercidicus NBRC 13090.

BACKGROUND: Tubercidin (TBN), an adenosine analog with potent antimycobacteria and antitumor bioactivities, highlights an intriguing structure, in which a 7-deazapurine core is linked to the ribose moiety by an N-glycosidic bond. However, the molecular logic underlying the biosynthesis of this antibiotic has remained poorly understood. RESULTS: Here, we report the discovery and characterization of the TBN biosynthetic pathway from Streptomyces tubercidicus NBRC 13090 via reconstitution of its production in a heterologous host. We demonstrated that TubE specifically utilizes phosphoribosylpyrophosphate and 7-carboxy-7-deazaguanine for the precise construction of the deazapurine nucleoside scaffold. Moreover, we provided biochemical evidence that TubD functions as an NADPH-dependent reductase, catalyzing irreversible reductive deamination. Finally, we verified that TubG acts as a Nudix hydrolase, preferring Co2+ for the maintenance of maximal activity, and is responsible for the tailoring hydrolysis step leading to TBN. CONCLUSIONS: These findings lay a foundation for the rational generation of TBN analogs through synthetic biology strategy, and also open the way for the target-directed search of TBN-related antibiotics.

Structural Basis for the KlenTaq DNA Polymerase Catalysed Incorporation of Alkene- versus Alkyne-Modified Nucleotides.

Efficient incorporation of modified nucleotides by DNA polymerases is essential for many cutting-edge biomolecular technologies. The present study compares the acceptance of either alkene- or alkyne-modified nucleotides by KlenTaq DNA polymerase and provides structural insights into how 7-deaza-adenosine and deoxyuridine with attached alkene-modifications are incorporated into the growing DNA strand. Thereby, we identified modified nucleotides that prove to be superior substrates for KlenTaq DNA polymerase compared with their natural analogues. The knowledge can be used to guide future design of functionalized nucleotide building blocks.

Nucleoside analog studies indicate mechanistic differences between RNA-editing adenosine deaminases.

Adenosine deaminases acting on RNA (ADAR1 and ADAR2) are human RNA-editing adenosine deaminases responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. Since inosine is recognized during translation as guanosine, this often results in the expression of protein sequences different from those encoded in the genome. While our knowledge of the ADAR2 structure and catalytic mechanism has grown over the years, our knowledge of ADAR1 has lagged. This is due, at least in part, to the lack of well defined, small RNA substrates useful for mechanistic studies of ADAR1. Here, we describe an ADAR1 substrate RNA that can be prepared by a combination of chemical synthesis and enzymatic ligation. Incorporation of adenosine analogs into this RNA and analysis of the rate of ADAR1 catalyzed deamination revealed similarities and differences in the way the ADARs recognize the edited nucleotide. Importantly, ADAR1 is more dependent than ADAR2 on the presence of N7 in the edited base. This difference between ADAR1 and ADAR2 appears to be dependent on the identity of a single amino acid residue near the active site. Thus, this work provides an important starting point in defining mechanistic differences between two functionally distinct human RNA editing ADARs.

Structure and functional characterization of the atypical human kinase haspin.

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.

A single adenosine with a neutral pKa in the ribosomal peptidyl transferase center.

Biochemical and crystallographic evidence suggests that 23S ribosomal RNA (rRNA) is the catalyst of peptide bond formation. To explore the mechanism of this reaction, we screened for nucleotides in Escherichia coli 23S rRNA that may have a perturbed pKa (where Ka is the acid constant) based on the pH dependence of dimethylsulfate modification. A single universally conserved A (number 2451) within the central loop of domain V has a near neutral pKa of 7.6 +/- 0.2, which is about the same as that reported for the peptidyl transferase reaction. In vivo mutational analysis of this nucleotide indicates that it has an essential role in ribosomal function. These results are consistent with a mechanism wherein the nucleotide base of A2451 serves as a general acid base during peptide bond formation.

A promiscuous kinase inhibitor delineates the conspicuous structural features of protein kinase CK2a1.

Protein kinase CK2a1 is a serine/threonine kinase that plays a crucial role in the growth, proliferation and survival of cells and is a well known target for tumour and glomerulonephritis therapies. Here, the crystal structure of the kinase domain of CK2a1 complexed with 5-iodotubercidin (5IOD), an ATP-mimetic inhibitor, was determined at 1.78 Šresolution. The structure shows distinct structural features and, in combination with a comparison of the crystal structures of five off-target kinases complexed with 5IOD, provides valuable information for the development of highly selective inhibitors.

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2'-deoxyadenosine.

Standard nucleobases all present electron density as an unshared pair of electrons to the minor groove of the double helix. Many heterocycles supporting artificial genetic systems lack this electron pair. To determine how different DNA polymerases use the pair as a substrate specificity determinant, three Family A polymerases, three Family B polymerases and three reverse transcriptases were examined for their ability to handle 3-deaza-2'-deoxyadenosine (c3dA), an analog of 2'-deoxyadenosine lacking the minor groove electron pair. Different polymerases differed widely in their interaction with c3dA. Most notably, Family A and Family B polymerases differed in their use of this interaction to exploit their exonuclease activities. Significant differences were also found within polymerase families. This plasticity in polymerase behavior is encouraging to those wishing to develop a synthetic biology based on artificial genetic systems. The differences also suggest either that Family A and Family B polymerases do not share a common ancestor, that minor groove contact was not used by that ancestor functionally or that this contact was not sufficiently critical to fitness to have been conserved as the polymerase families diverged. Each interpretation is significant for understanding the planetary biology of polymerases.

Initial rate kinetics of the transport of adenosine and 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin) in cultured cells.

A procedure is described for determining early time courses of nucleoside uptake by cultured cells in suspension. Replicate samples of cell suspensions were exposed to medium containing 3H-nucleosides for brief intervals (sec) ended by addition of nitrobenzylthioinosine, a potent inhibitor of nucleoside transport that terminated nucleoside uptake virtually instantaneously. Time courses of nucleoside uptake were constructed from the cellular content of nucleoside acquired by the replicate samples during graded intervals of exposure to the labeled permeant. Such time courses were definitive of cellular uptake of nucleosides during the first few sec of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(beta-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin). Defining initial rates of nucleoside uptake as rates of inward transport, relationships between transport rates and extracellular concentrations of these permeants were evaluated in HeLa cells and in two cultured lines of mouse lymphoma L5178Y cells that differ in their abilities to phosphorylate adenosine and tubercidin. Transport rates for these permeants were similar in the two L5178Y cell types and were saturable in the 3 cell lines with Km values between 14 and 38 microM. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.

Study of the cytotoxicity and metabolism of 4-amino-3-carboxamido-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine using inhibitors of adenosine kinase and adenosine deaminase.

The greater in vivo antitumor activity of 4-amino-3-carboxamido-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (APPCR) in comparison to the parent compound 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (APPR) may involve intrinsic differences in the effects of these two compounds on cells, as well as in their metabolisms. In studies with L1210 cells in vitro, growth inhibition by APPCR for 36 hr or more was found to be cytocidal, while growth inhibition by APPR was cytostatic in that a substantial fraction of the cells survived 36 or 72 hr of exposure to growth-inhibiting concentrations of APPR. It appears that this difference in the cellular effects of APPCR and APPR is not related to differences in the requirement for activation by phosphorylation or in susceptibility to inactivation by deamination. However, deamination may limit the effectiveness of APPR in vivo since it is a substrate for adenosine deaminase, while deamination of APPCR is not detectable.

Resistance to 9-beta-D-arabinofuranosyladenine in cultured leukemia L 1210 cells.

A cultured line of L1210 leukemia cells, designated L1210/ara-A, was selected for resistance to 9-beta-D-arabinofuranosyladenine (ara-A) by a series of 72-hr exposures to increasing concentrations of ara-A in the presence of 1 microM deoxycoformycin. Cells of the resistant line were about one-tenth as sensitive as were cells of the parent line to the effects of ara-A on proliferation, viability, and tumorigenicity. Cross-resistance, as determined by comparison of drug effects on rates of proliferation of L1210/C2 and L1210/ara-A cells, was seen with adenosine, deoxyadenosine, methylmercaptopurine ribonucleoside, tubercidin, and cordycepin but not with 1-beta-D-arabinofuranosylcytosine or with 9-beta-D-arabinofuranosyl-2-fluoroadenine. The levels of resistance to methylmercaptopurine ribonucleoside, cordycepin, and tubercidin were considerably greater than that seen with ara-A itself. L1210/C2 and L1210/ara-A cells were compared with respect to the effects of ara-A on cell size distributions, DNA distributions, labeling indices, and apparent rates of DNA synthesis, and the differences seen were consistent with inhibition of DNA synthesis and unbalanced growth as the major mechanism of ara-A cytotoxicity. The decreased sensitivity of DNA synthesis in L1210/ara-A cells treated with ara-A, relative to L1210/C2 cells, was due to reduced intracellular accumulation of ara-A phosphates in the resistant line. Phosphorylation of ara-A, adenosine, and tubercidin, but not deoxyadenosine or deoxycytidine, was greatly reduced in intact L1210/ara-A cells, relative to L1210/C2 cells, and adenosine kinase activity in extracts of L1210/ara-A cells was negligible. Resistance to ara-A, and cross-resistance to tubercidin, methylmercaptopurine ribonucleoside, and cordycepin is attributed to loss of adenosine kinase activity.

Adenosine receptors and modulation of natural killer cell activity by purine nucleosides.

Natural killer (NK) cell activity is inhibited by the adenosine analogue tubercidin (Tub) and stimulated by the deoxyadenosine analogue 2-fluoro-1-beta-D-arabinofuranosyladenine 5'-monophosphate (F-ara-AMP) in the spleen lymphocytes from mice treated with the drugs in vivo (T. Priebe et al., Cancer Res., 48:4799, 1988). The present report demonstrates that the inhibition by Tub and stimulation by F-ara-AMP of NK cell activity are readily demonstrable in murine and human lymphocytes exposed to the drugs in vitro. In mouse spleen lymphocytes, NK cell activity is also inhibited by adenosine receptor A2 agonists, whereas potent A1 receptor agonists are more effective stimulators. Inhibition produced by adenosine, deoxyadenosine, and adenosine receptor agonists, but not by Tub, is partially prevented by the adenosine receptor antagonist 1,3-dipropyl-8-phenylxanthine amine congener. Agents that stimulate NK cell activity (deoxyadenosine, A1 receptor agonists, F-ara-AMP) do not increase further the 1.5-fold enhancement produced by a 10(-6) M concentration of 1,3-dipropyl-8-phenylxanthine amine congener. The nucleoside transport inhibitor, p-nitrobenzylthioinosine 5'-monophosphate, has no effect on NK cell activity or intracellular ribonucleotide pools; however, it partially prevents Tub 5'-triphosphate formation, ATP depletion, and NK cell inhibition in mouse spleen cells treated with Tub. p-Nitrobenzylthioinosine 5'-monophosphate also partially prevents the F-ara-AMP stimulation of NK cell activity, but it does not influence the effects of adenosine or deoxyadenosine. The results obtained with the adenosine receptor agonists suggest roles for both A1 and A2 receptors in regulating murine NK cell activity. Tub inhibition of NK cell activity does not appear to involve adenosine receptors; however, inhibition by the other agents may be mediated via an A2 receptor (stimulatory for adenylyl cyclase). Since p-nitrobenzylthioinosine 5'-monophosphate inhibited the stimulation of NK cell activity by F-ara-AMP, this stimulation may occur via an intracellular "P" site (inhibitory to adenylyl cyclase).

Transport and metabolism of 9-beta-D-arabinofuranosylguanine in a human T-lymphoblastoid cell line: nitrobenzylthioinosine-sensitive and -insensitive influx.

Nitrobenzylthioinosine (NBMPR), dipyridamole, and dilazep, potent inhibitors of nucleoside transport, were found to be ineffective in preventing 9-beta-D-arabinofuranosylguanine (ara-G)-induced inhibition of MOLT 4 and CCRF CEM cell growth, ara-G (2.0 microM) was metabolized to 9-beta-D-arabinofuranosylguanine 5'-triphosphate in MOLT 4 cells, and the levels of this metabolite were not affected by the presence of 5.0 microM NBMPR in the incubation medium. Permeation of the MOLT 4 cell membrane by ara-G occurred primarily by means of the NBMPR-sensitive nucleoside transport system. However, a residual transport component accounting for 10-20% of the total transport activity was demonstrated in the presence of NBMPR. This component was inhibited by adenine and hypoxanthine but not by dilazep, dipyridamole, or other nucleosides. In contrast, inhibitors of nucleoside transport readily reversed the cytotoxic effect of 7-deazaadenosine (tubercidin) in both MOLT 4 and CCRF CEM cells. The levels of tubercidin 5'-triphosphate formed from 2.0 microM tubercidin in MOLT 4 cells were reduced by 80% in the presence of 5.0 microM NBMPR. The influx of tubercidin into MOLT 4 cells was found to occur primarily by means of the NBMPR-sensitive nucleoside transport system. This same system mediated the transport of ara-G into human erythrocytes.

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action.

7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.

5-methylthioribose 1-phosphate: a product of partially purified, rat liver 5'-methylthioadenosine phosphorylase activity.

5'-Methylthioadenosine phosphorylase from rat liver has been purified 112-fold. A molecular weight of 90 000 for the enzyme was estimated from gel filtration on Sephadex G-150. The Km for 5'-methylthioadenosine was 4.7 . 10(-7) M, while the Km for phosphate was 2 . 10(-4) M. The products of the reaction were isolated and identified as adenine and 5-methylthioribose 1-phosphate. In addition to 5'-methylthioadenosine the nucleoside analogues 5'-ethylthioadenosine and 5'-n-propylthioadenosine also served as substrates for the enzyme. The 7-deaza analogue 5'-methylthiotubercidin was found to be an inhibitor of the reaction, but was inactive as a substrate.

Liposome-cell interactions. A study of the interactions of liposomes containing entrapped anti-cancer drugs with the EMT6, S49 and AE1 (transport-deficient) cell lines.

A study has been made to determine if the cytotoxicity observed when cells in culture were exposed to liposome-entrapped cytotoxic drugs was liposome mediated or resulted from leakage of drug from the liposomes with subsequent uptake of free drugs by the cells. In preliminary experiments with the EMT6 cell line in monolayer culture, the cytotoxicity observed when the cells were exposed to a range of concentrations of liposome-entrapped methotrexate, actinomycin D and cytosine arabinoside for a variety of liposome compositions was somewhat less than that observed when the cells were exposed to similar concentrations of free drug. We suspected that the cytotoxicity was mediated via uptake of free drug leaked from liposomes. This was confirmed in experiments involving the EMT6 and S49 cell lines in monolayer or suspension culture, respectively, in the absence and presence of the nucleoside transport inhibitor, 6-(4-nitrobenzyl)thio)-9-beta-D-ribofuranosylpurine. Additional experiments were performed in a transport-deficient mutant of the S49 cell line, the AE1 cell line. No evidence for liposome-mediated cell death could be found in these cell lines when tubercidin 5'-monophosphate was entrapped in either large or small unilamellar liposomes composed of egg phosphatidylcholine/cholesterol (2 : 1), bovine brain phosphatidylserine/egg phosphatidylcholine/cholesterol (8 : 2 : 5) or egg phosphatidylcholine/stearylamine/cholesterol (10 : 1 : 5). Considerable toxicity due to empty liposomes of a variety of compositions was observed in the S49 cell line at high lipid concentrations.

Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax.

The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.