Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate

Ostermann, Nils; Schlichting, Ilme; Brundiers, R.; Konrad, Manfred; Reinstein, Jochen; Veit, T.; Goody, Roger S.; Lavie, A.

doi:10.1016/s0969-2126(00)00149-0

Cited by 101 publications

(132 citation statements)

References 27 publications

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“…In addition, the LID region, which is another phosphoryl donor binding site, was also found. However, the amount of conservation was much lower, as has been reported previously in humans, yeast and E. coli (Lavie et al, 1998;Ostermann et al, 2000). These results indicated that TMK-a and TMK-b have conserved functional motifs in their primary sequence, and suggest that they may function as TMK enzymes.…”

Section: Tmk Genes Have Conserved Functional Motifssupporting

confidence: 70%

“…TMK enzymes are globular dimeric proteins with a folding pattern similar to that of nucleoside monophosphate kinases (Ostermann et al, 2000), which possess three loops crucial for enzyme activity: the phosphate-binding motif at the Nterminus (P-loop), the nucleoside-monophosphate-binding domain, and the region that covers part of the P-loop upon substrate binding (the LID domain).…”

Section: Introductionmentioning

confidence: 99%

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Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

et al. 2003

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Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the onion yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.

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Section: Tmk Genes Have Conserved Functional Motifssupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

et al. 2003

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“…Deoxynocleotide kinase (31) also exhibits such a motif but the functional test of the comparable arginines has only been reported in this work on PMK. Ostermann et al (30) have proposed that, for thymidylate kinase, a basic residue near a Walker B motif bridges phosphoryl donor and acceptor substrates. In such a case, the basic side chain (e.g.…”

Section: Discussionmentioning

confidence: 99%

Functional Evaluation of Conserved Basic Residues in Human Phosphomevalonate Kinase

Herdendorf

Miziorko

2007

Biochemistry

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Phosphomevalonate kinase (PMK) catalyzes the cation dependent reaction of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, a key step in the mevalonate pathway for isoprenoid/sterol biosynthesis. Animal PMK proteins belong to the nucleoside monophosphate (NMP) kinase family. For many NMP kinases, multiple basic residues contribute to the neutralization of the negatively charged pentacoordinate phosphate reaction intermediate. Loss of basicity can result in catalytically impaired enzymes. Based on this precedent, conserved basic residues of human PMK have been mutated and purified forms of the mutated proteins have been kinetically and biophysically characterized. K48M and R73M mutants exhibit diminished V max values in both reaction directions (>1000-fold) with only slight K m perturbations (<10-fold). In both forward and reverse reactions, R110M exhibits a large (>10,000-fold) specific activity diminution. R111M exhibits substantially inflated K m values for mevalonate 5-phosphate and mevalonate 5-diphosphate (60 and 30-fold, respectively) as well as decreases (50-fold (forward) and 85-fold (reverse)) in V max . R84M also exhibits inflated K m values (50 and 33-fold for mevalonate 5-phosphate and mevalonate 5-diphosphate, respectively). The K i values for R111M and R84M product inhibition by mevalonate 5-diphosphate are inflated by 45-and 63-fold; effects are comparable to the 30-and 38-fold inflations in K m for mevalonate 5-diphosphate. R141M exhibits little perturbation in V max (14-fold (forward) and 10-fold (reverse)) but has inflated K m values for ATP and ADP (48 and 136-fold, respectively). The K d of ATP for R141M, determined by changes in tryptophan fluorescence, is inflated 27-fold compared to wt PMK. These data suggest that R110 is important to PMK catalysis, which is also influenced by K48 and R73. R111 and R84 contribute to binding of mevalonate 5-phosphate and R141 to binding of ATP.Phosphomevalonate kinase (PMK 1 ; EC 2.7.4.2) catalyzes the reversible ATP dependent phosphorylation of mevalonate 5-phosphate to produce mevalonate 5-diphosphate and ADP.The reaction represents a key step in the mevalonic acid mediated biosynthesis of isopentenyl diphosphate and other polyisoprenoid metabolites. Disruption of the PMK encoding gene in yeast (1) has demonstrated the essentiality of this enzyme. Enzyme activity was documented in pig liver (2) and initial characterization work employed the purified porcine enzyme (3,4). The sequence of human PMK has been deduced (5) and expression of a recombinant GST- † This work was supported in part by NIDDK

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“…Subsequent work with the human TMPK supported the conclusions derived from the yeast TMPK structures [17]. Importantly, the human TMPK structures are of much higher resolution than the yeast TMPK structures, and include a nucleotide molecule bound to the phosphoryl donor site (either ADP or the ATP analog adenosine β,γ-imido-5'-triphosphate (AppNHp)) and the essential magnesium ion.…”

Section: (1) Aztmpmentioning

confidence: 81%

“…The importance of the sugar pucker for TMPK-dependent phosphorylation is evident from the results obtained with d4TMP and ddTMP. Since the analog's phosphate group must be able to change its conformation to interact Arg97 and with Arg45 [17], it is possible that by locking the sugar pucker into a rigid conformation, and hence the position of the phosphate group, this required mobility was lost.…”

Section: (C) Acyclic-sugar and Conformationally-locked Thymidine Analogsmentioning

confidence: 99%

Structural Requirements for Efficient Phosphorylation of Nucleotide Analogs by Human Thymidylate Kinase

Lavie

Konrad

2004

mrmc

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Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required for the pharmacological activity of these compounds in the chemotherapeutic treatment of viral infections and cancer. Human thymidylate kinase (TMPK), apart from its essential physiological role in the biosynthesis of TTP, is also required for the activation of thymidine analogs, such as the clinically used anti-HIV prodrugs AZT and d4T. This enzyme is rate determining in the three-step cascade of AZT phosphorylation. Our structural work on human, yeast and E. coli TMPKs, in conjunction with sequence homology analyses and biochemical data, has demonstrated that three loops are crucial for the function of this enzyme: the first is the highly conserved P-loop motif, which binds and positions the phosphoryl groups of ATP, the second critical loop contains the DR(Y/H) motif that supplies a catalytic arginine and is also important for the binding and positioning of the magnesium ion complexed to ATP, and the third loop is the so-called Lid-region that is a flexible stretch which closes on ATP when it binds. Modifications of the sugar moieties of nucleoside monophosphates are shown to exert drastic effects on the enzyme's conformation and, thus, reduced activity. Our structural work on several TMPKs has formed the basis for generating mutants of human TMPK that are about 100 times more efficient in phosphorylating AZTMP. These enzyme variants could potentially be introduced into HIV-targeted cells in order to significantly improve AZT's antiviral activity.

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Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate

Cited by 101 publications

References 27 publications

Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

Functional Evaluation of Conserved Basic Residues in Human Phosphomevalonate Kinase

Structural Requirements for Efficient Phosphorylation of Nucleotide Analogs by Human Thymidylate Kinase

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