Nucleoside and deoxyribonucleoside kinases

These types of enzymes carry out the 5'-phosphorylation of deoxy- and ribonucleosides and related analogs using a nucleoside triphosphate (usually ATP) as phosphate donor in the presence of Mg2+ ions (Figure 2.2). A drawback for these types of enzymes in synthetic reactions is that they usually have a rather narrow specificity and thus are highly selective for certain types of nucleosides. The introduction of adeosine kinase (E.C. 2.7.1.20) as a synthetic tool has been pioneered by Whitesides and colleagues11 and adenosine has been converted to AMP using polyacrylamide cross linked commercially available adenosine kinase (Figure 2.2).

Usually, the reactions were carried out in a mixture also containing adenylate kinase and acetate kinase with ATP, ADP, AMP, Mg2+ acetate, and

Nucleosides (d)NK:s

Deoxynucleosides Analogs

PK/PEP;AcK/AcP;CrK/CrP

NDPK

PK/PEP;AcK/AcP;CrK/CrP

NDPK

Figure 2.2 Overview of NTP synthesis with nucleoside or deoxynucleoside kinase followed by nucleotide kinases as described in the legend to Figure 2.1.

the reducing agent dithiotreitol (DTT); the overall yield of the final product ATP was 98%.11 A similar approach based on crude preparations of yeast adenosine kinase and adenylate kinase for the continues synthesis of ATP has been described.12 The rationale for the coupled reactions will be described in the following sections. Several crude preparations or partially purified preparations have been used, e.g., uridine-cytidine kinase to phos-phorylate 5-azacytidine to 5-aza-CMP in small scale (at pH 6.6 to stabilize the analog) with about 12% yields.13

Adenosine (AK) regulates the extracellular adenosine and intracellular adenylate concentrations and inhibition of the enzyme elevate adenosine to levels that activate nearby adenosine receptors and produce a wide variety of physiological activities. The structure of the human adenosine kinase has been determined.14 The overall structure is similar to the structure of riboki-nase from Escherichia coli, which belongs to the family of carbohydrate kinases.

Two molecules of adenosine were present in the initial AK crystals with one adenosine molecule located in a site that matches the ribose site in ribokinase and probably represents the substrate-binding site. The second adenosine site probably represents the ATP site. An Mg2+ ion-binding site is observed in a trough between the two adenosine sites. The structure of the active site is consistent with the observed substrate specificity. The specificity of AK is relatively restricted to adenosine, but the enzyme can accept deoxy-adenosine as well as certain analogs (see below) with much lower, but still significant, activity.15 Uridine-cytidine kinase, which belongs to the same enzyme family, has been used for synthetic purposes,13 but only to a very limited extent.

Some recent synthetic applications of the broadly specific deoxynucleo-side kinases from Drosophila melanogaster (DmdNK) and plants, e.g., tomato-dNK, will be presented here (Figure 2.2). DmdNK has recently been shown to have considerable potential as a more general and efficient phos-phorylating agent for a large variety of nucleosides and nucleoside ana-logs.1617 The plant dNK enzymes have recently been cloned and characterized and shown to belong to the same family as the DmdNK as well as the mammalian deoxycytidine kinase/deoxyguanosine kinase family.19 The dNK from tomato (Lycopericum escultentum) may be of special interest for biosynthetic applications because it apparently can also carry out the phos-phorylation of nucleoside monophosphates, although at a very low rate.18 Further development of this class of enzymes may lead to new tools of value for synthesis of nucleotides.

The DmdNK enzyme has some major advantages compared to other deoxynucleoside kinases in that it has not only a very broad acceptance for different nucleoside substrates but also a high catalytic rate; it is stable even at higher temperatures and requires no reducing agents in order to maintain full activity. The synthesis of dCMP, dGMP, or dAMP occurs with approximately 80% yields in 60 h using ATP, Mg2+, and creatine kinase as phosphate donor regenerating system. The Drosophila enzyme has been used for commercial production of dideoxynucleotide analogs.17

The structure of DmdNK was recently determined19 and the results demonstrated that it belongs to the nucleotide/deoxynucleoside kinase family, which also includes HSV1-TK. These enzyme structures consist of a dimer of two 31-kDa subunits, each with an alpha/beta-structure made of 9 to 10 helices and five beta-sheets with an active site cleft in which a nucleoside-binding domain and the ATP-binding loop are key features. This latter loop is formed by a strand-turn-helix that forms a large anion hole that coordinates the phosphate group of the phosphate donors.

This type of ATP-binding structure is found in all nucleoside/nucleotide kinases studied so far. On top of the active site, a helix containing basic amino acids forms the s c lid region. The length and size of the lid region varies in different nucleoside/nucleotide kinases, but there is always this kind of function because it allows shielding of the active site in order to limit the access for water molecules that otherwise will compete with the 5'-OH group of the nucleosides and result in the transfer reaction. The capacity of DmdNK to phosphorylate many different nucleoside and nucleoside analogs at high rates has been described as well as many important structure-function relationships.16-19

Another member of this enzyme family that has been in focus for pharmacological investigations for two decades is herpes virus 1 thymidine kinase. The enzyme is a dimer of two 44-kDa subunits; its specificity towards nucleosides is very broad and many acyclic analogs such as acyclovir and ganciclovir are efficiently phosphorylated.7820 This is the underlying mechanism for the antiviral selectivity of most antiherpes drugs. The literature on the structure and activity of this enzyme in complex with many analogs20 is extensive.

The herpes virus type 1 enzyme belongs to a different enzyme family than the adenosine kinases do, but shows high structural similarity with the insect and mammalian deoxycytidine/deoxyguanosine kinases as described earlier.19 Herpes TK enzymes have recently received much attention in gene therapy applications.21 This is again due to its broad substrate specificity, which includes L nucleosides and many other antiviral analogs not phos-phorylated by the cellular deoxynucleoside kinases. Therefore, treatment with such analogs can be made very selective with minimal toxic side effect to nontransfected cells. Nevertheless, so far these viral enzymes have not been extensively used for synthetic approaches.

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