The enzyme, found in prokaryotes and eukaryotes, phosphorylates both uridine and cytidine to their monophosphate forms. The enzyme from Escherichia coli prefers GTP to ATP. The human enzyme also catalyses the phosphorylation of several cytotoxic ribonucleoside analogs. cf. EC 2.7.1.213, cytidine kinase.
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SYSTEMATIC NAME
IUBMB Comments
ATP:uridine/cytidine 5'-phosphotransferase
The enzyme, found in prokaryotes and eukaryotes, phosphorylates both uridine and cytidine to their monophosphate forms. The enzyme from Escherichia coli prefers GTP to ATP. The human enzyme also catalyses the phosphorylation of several cytotoxic ribonucleoside analogs. cf. EC 2.7.1.213, cytidine kinase.
UCK from Thermus thermophilus HB8 loses catalytic activity on uridine due to lack of a substrate binding ability and possesses an unusual amino acid, i.e. tyrosine 93 (Tyr93) at the binding site, whereas histidine (His) is located in the other UCKs. Mutagenesis experiments reveal that a replacement of Tyr93 by His or glutamine (Gln) recovers catalytic activity on uridine
uridine-cytidine kinase (UCK) is one of the enzymes in the nucleoside salvage pathway. UCK generally converts both cytidine and uridine to nucleoside monophosphate using ATP as the phosphate donor, but the UCK of Thermus thermophilus HB8 (ttCK) phosphorylates only cytidine. This cytidine-restricted activity is thought to depend on residue Tyr93
mechanism underlying nucleoside specificity, overview. The histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK, due to the naturally occuring polymorphism Y93, the UCK homologue ttCK of Thermus thermophilus HB8 has, unlike other UCKs, a substrate specificity towards only cytidine and shows no inhibition by UTP, uridine does not bind to ttCK as substrate. Replacement of Tyr93 by histidine or glutamine endows ttCK with phosphorylation activity toward uridine
mechanism underlying nucleoside specificity, overview. The histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK, due to the naturally occuring polymorphism Y93, the UCK homologue ttCK of Thermus thermophilus HB8 has, unlike other UCKs, a substrate specificity towards only cytidine and shows no inhibition by UTP, uridine does not bind to ttCK as substrate. Replacement of Tyr93 by histidine or glutamine endows ttCK with phosphorylation activity toward uridine
molecular dynamics simulations on the wild-type Thermus thermophilus enzyme, two mutant ttCKs, and a human UCK bound to cytidine and three protonation forms of uridine to elucidate their substrate specificity, overview. Three residues, Tyr88, Tyr/His/Gln93 and Arg152 in ttCKs, are important for recognizing the substrates. Arg152 contributes to induce a closed form of the binding site to retain the substrate, and the N3 atom of uridine needs to be deprotonated. Although Tyr88 tightly binds cytidine, it does not sufficiently bind uridine because of lack of the hydrogen bonding. His/Gln93 complements the interaction of Tyr88 and raises the affinity of ttCK to uridine. The crucial distinction between Tyr and His or Gln is a role in the hydrogen-bonding network. Therefore, the ability to form both hydrogen-bonding donor and acceptor is required to bind both uridine and cytidine. Residue interactions and kinetics. Tyr59 and Phe90 form Pi-Pi stackings and CH-Pi interaction with cytidine, and Ile113 interacts with cytidine via a hydrophobic interaction. Tyr88, His93, and Arg152 form hydrogen bonds with the cytidine base moiety, and Asp60 and Arg142 anchor the ribose moiety, role of His/Gln93 in complementing the interaction between Tyr88 and the substrate
uridine phosphorylation activity commonly depends on a single residue in the UCK family. Molecular docking analysis. Structure comparison of Thermus thermophilus enzyme ttCK and human enzyme hsUCK2
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complex with cytidine and beta,gamma-methyleneadenosine 5'-triphosphate, sitting drop vapor diffusion method, using 0.1 M NaCl, 0.1 M bicine, pH 9.0, and 20% (w/v) polyethylene glycol monomethyl ether 550
purified recombinant enzyme H93Y variant ttCK free or incomplex with CMP, sitting-drop vapor diffusion method, mixing of 500 nl of 25 mg/ml protein, with 1 mM CMP, 1 mM ADP, and 1 mM MgCl2 in case of the enzyme complex, with 500 nl well solution containing 67% 40% v/v isopropanol, 15% w/v PEG 8000, and 0.1 M imidazole, pH 6.5, and 33% distilled water for the free enzyme, and containing 50% w/v PEG 200, and 0.1 M Tris-HCl, pH 8.0, for the enzyme complex, 20°C, X-ray diffraction structure determination and analysis at 2.25 A resolution, molecular replacement
site-directed mutagenesis, the mutant enzyme shows restored activity on uridine in contrast to the wild-type enzyme. The mutant has higher binding affinities to cytidine than the wild-type
site-directed mutagenesis, the mutant enzyme shows restored activity on uridine in contrast to the wild-type enzyme. The mutant has higher binding affinities to cytidine than the wild-type
the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK, due to the naturally occuring polymorphism Y93, the UCK homologue ttCK of Thermus thermophilus HB8 has, unlike other UCKs, a substrate specificity towards only cytidine and shows no inhibition by UTP, uridine does not bind to ttCK as substrate. Replacement of Tyr93 by histidine or glutamine endows ttCK with phosphorylation activity toward uridine
the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK, due to the naturally occuring polymorphism Y93, the UCK homologue ttCK of Thermus thermophilus HB8 has, unlike other UCKs, a substrate specificity towards only cytidine and shows no inhibition by UTP, uridine does not bind to ttCK as substrate. Replacement of Tyr93 by histidine or glutamine endows ttCK with phosphorylation activity toward uridine
recombinant ttCK from Escherichia coli strain Rosetta(DE3) by heat treatment at 60°C for 20 min, hydrophobic interaction chromatography, and gel filtration
recombinant wild-type and mutant enzymes from Escherichia coli strain Rosetta(DE3) by heat treatment at 70°C for 20 min, followed by hydrophobic interaction chromatography, and two different steps of gel filtration