Enantioselectivity of human AMP, dTMP and UMP-CMP kinases

Alexandre, Julie Anne Christine; Roy, Béatrice; Topalis, Dimitrios; Pochet, Sylvie; Philouze, Christian; Deville‐Bonne, Dominique

doi:10.1093/nar/gkm479

Cited by 18 publications

(29 citation statements)

References 43 publications

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“…Vacc-TMPK was more sensitive to dT than was the human enzyme (Table 1). This agrees with the previous determination of dT affinity by a competition assay using fluorescent TDP analog as a probe: K D dT was Ϸ2 M for Vacc-TMPK and 40 M for the human enzyme (20,28). The better affinity of dT for the vaccinia enzyme is probably due to the P-loop Asp-13 maintained in closed conformation by H-bonding to Tyr-101, a residue replaced by Phe in hTMPK.…”

Section: Resultssupporting

confidence: 91%

Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy

Caillat

Topalis

Agrofoglio

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Unlike most DNA viruses, poxviruses replicate in the cytoplasm of host cells. They encode enzymes needed for genome replication and transcription, including their own thymidine and thymidylate kinases. Some herpes viruses encode only 1 enzyme catalyzing both reactions, a peculiarity used for prodrug activation to obtain maximum specificity. We have solved the crystal structures of vaccinia virus thymidylate kinase bound to TDP or brivudin monophosphate. Although the viral and human enzymes have similar sequences (42% identity), they differ in their homodimeric association and active-site geometry. The vaccinia TMP kinase dimer arrangement is orthogonal and not antiparallel as in human enzyme. This different monomer orientation is related to the presence of a canal connecting the edge of the dimer interface to the TMP base binding pocket. Consequently, the pox enzyme accommodates nucleotides with bulkier bases, like brivudin monophosphate and dGMP; these are efficiently phosphorylated and stabilize the enzyme. The brivudin monophosphate-bound structure explains the structural basis for this specificity, opening the way to the rational development of specific antipox agents that may also be suitable for poxvirus TMP kinase gene-based chemotherapy of cancer.BVdU ͉ dihalovinyl-dU ͉ dimerization mode ͉ TMP kinase ͉ vaccinia virus

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Section: Resultssupporting

confidence: 91%

Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy

Caillat

Topalis

Agrofoglio

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…). These values of catalytic efficiencies obtained with these two direct detection methods are close to those previously reported in the literature[6,43]. Therefore, FIA-HRMS methodology does not bring…”

supporting

confidence: 89%

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry

Ferey

Silva

Colas

et al. 2019

Analytica Chimica Acta

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Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

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“… [a] Kinetic parameters of AMP, PMPA and PMEA are taken from the report by Alexandre et al 27. and Topalis et al.…”

Section: Resultsmentioning

confidence: 99%

5′,6′‐Nucleoside Phosphonate Analogues Architecture: Synthesis and Comparative Evaluation towards Metabolic Enzymes

et al. 2011

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Nucleoside phosphonates have been designed as stable 5'-mononucleotide mimics and are nowadays considered a potent class of antiviral agents. Within cells, they must be metabolised to the corresponding diphosphate to exert their biological activity. In this process, the first phosphorylation step, catalysed by nucleoside monophosphate kinases (NMP kinases), has been proposed as a bottleneck. Herein, we report the synthesis of a series of ribonucleoside phosphonate derivatives isosteric to 5'-mononucleotides, with different degrees of flexibility within the 5',6'-C-C bond, as well as different polarities, through the introduction of hydroxy groups. The influence of these modifications on the capacity of the compounds to act as substrates for appropriate human NMP kinases, involved in nucleic acids metabolism, has been investigated. Low flexibility, as well as an absence of hydroxy groups within the ribose-phosphorus architecture, is critical for efficient phosphotransfer. Among the series of pyrimidine analogues, one derivative was shown to be phosphorylated by human UMP-CMP kinase, with rates similar to those of dUMP and even better than dCMP.

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Enantioselectivity of human AMP, dTMP and UMP-CMP kinases

Cited by 18 publications

References 43 publications

Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy

Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry

5′,6′‐Nucleoside Phosphonate Analogues Architecture: Synthesis and Comparative Evaluation towards Metabolic Enzymes

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