Nucleoside Monophosphate Kinase Research Articles

CMPK2 Promotes CD4+ T Cell Activation and Apoptosis through Modulation of Mitochondrial Dysfunction in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by abnormal autoantibodies, immune complex deposition, and tissue inflammation. Despite extensive research, the exact etiology and progression of SLE remain elusive. Cytidine/uridine monophosphate kinase 2 (CMPK2), a mitochondrial nucleoside monophosphate kinase, has garnered attention for its potential involvement in the development of various diseases, including SLE, where it has been observed to be dysregulated in affected individuals. However, the specific involvement of CMPK2 in the pathogenesis of SLE remains unclear. This study aims to clarify the expression level of CMPK2 in SLE CD4+ T cells and explore its impact on CD4+ T cells. The expression levels of the CMPK2 gene and the corresponding CMPK2 protein in CD4+ T cells of SLE patients were quantified using RT-qPCR and Western blot, respectively. Immunofluorescence and RT-qPCR were used to assess the mitochondrial function of SLE CD4+ T cells. Flow cytometry was used to assess CD4+ T cell activation and apoptosis levels. The impact of CMPK2 on CD4+ T cells was investigated by gene transfection experiment. We found that CMPK2 was significantly upregulated in SLE CD4+ T cells at both gene and protein levels. These cells demonstrated aberrant mitochondrial function, as evidenced by elevated mitochondrial reactive oxygen species (mtROS) levels, mitochondrial membrane potential, and mitochondrial DNA (mtDNA) copy number. Flow cytometry revealed a notable increase in both apoptosis and activation levels of CD4+ T cells in SLE patients. Gene transfection experiments showed that suppressing CMPK2 led to a significant improvement in these conditions. These findings suggest that CMPK2 may be involved in the pathogenesis of SLE by regulating mitochondrial dysfunction in CD4+ T cells and thus affecting CD4+ T cell activation and apoptosis. Our study may provide a new target for the treatment of SLE.

UMP-CMP kinase 2 gene expression in macrophages is dependent on the IRF3-IFNAR signaling axis.

Toll-like receptors (TLRs) are highly-conserved pattern recognition receptors that mediate innate immune responses to invading pathogens and endogenous danger signals released from damaged and dying cells. Activation of TLRs trigger downstream signaling cascades, that culminate in the activation of interferon regulatory factors (IRFs), which subsequently leads to type I interferon (IFN) response. In the current study, we sought to expand the scope of gene expression changes in THP1-derived macrophages upon TLR4 activation and to identify interferon-stimulated genes. RNA-seq analysis led to the identification of several known and novel differentially expressed genes, including CMPK2, particularly in association with type I IFN signaling. We performed an in-depth characterization of CMPK2 expression, a nucleoside monophosphate kinase that supplies intracellular UTP/CTP for nucleic acid synthesis in response to type I IFN signaling in macrophages. CMPK2 was significantly induced at both RNA and protein levels upon stimulation with TLR4 ligand—LPS and TLR3 ligand—Poly (I:C). Confocal microscopy and subcellular fractionation indicated CMPK2 localization in both cytoplasm and mitochondria of THP-1 macrophages. Furthermore, neutralizing antibody-based inhibition of IFNAR receptor in THP-1 cells and BMDMs derived from IFNAR KO and IRF3 KO knockout mice further revealed that CMPK2 expression is dependent on LPS/Poly (I:C) mediated IRF3- type I interferon signaling. In summary, our findings suggest that CMPK2 is a potential interferon-stimulated gene in THP-1 macrophages and that CMPK2 may facilitate IRF3- type I IFN-dependent anti-bacterial and anti-viral roles.

Multifaceted impact of a nucleoside monophosphate kinase on 5'-end-dependent mRNA degradation in bacteria.

A key pathway for mRNA degradation in bacterial cells begins with conversion of the initial 5′-terminal triphosphate to a monophosphate, a modification that renders transcripts more vulnerable to attack by ribonucleases whose affinity for monophosphorylated 5′ ends potentiates their catalytic efficacy. In Escherichia coli, the only proteins known to be important for controlling degradation via this pathway are the RNA pyrophosphohydrolase RppH, its heteromeric partner DapF, and the 5′-monophosphate-assisted endonucleases RNase E and RNase G. We have now identified the metabolic enzyme cytidylate kinase as another protein that affects rates of 5′-end-dependent mRNA degradation in E. coli. It does so by utilizing two distinct mechanisms to influence the 5′-terminal phosphorylation state of RNA, each dependent on the catalytic activity of cytidylate kinase and not its mere presence in cells. First, this enzyme acts in conjunction with DapF to stimulate the conversion of 5′ triphosphates to monophosphates by RppH. In addition, it suppresses the direct synthesis of monophosphorylated transcripts that begin with cytidine by reducing the cellular concentration of cytidine monophosphate, thereby disfavoring the 5′-terminal incorporation of this nucleotide by RNA polymerase during transcription initiation. Together, these findings suggest dual signaling pathways by which nucleotide metabolism can impact mRNA degradation in bacteria.

Modular Enzymatic Cascade Synthesis of Nucleotides Using a (d)ATP Regeneration System.

Nucleoside-5’-triphosphates (NTPs) and their analogs are building blocks of DNA and are important compounds in both pharmaceutical and molecular biology applications. Currently, commercially available base or sugar modified NTPs are mainly synthesized chemically. Since the chemical production of NTPs is time-consuming and generally inefficient, alternative approaches are under development. Here we present a simple, efficient and generalizable enzymatic synthesis method for the conversion of nucleosides to NTPs. Our one-pot method is modular, applicable to a wide range of natural and modified nucleotide products and accesses NTPs directly from cheap nucleoside precursors. Nucleoside kinases, nucleoside monophosphate (NMP) kinases and a nucleoside diphosphate (NDP) kinase were applied as biocatalysts. Enzymes with different substrate specificities were combined to produce derivatives of adenosine and cytidine triphosphate with conversions of 4 to 26%. The implementation of a (deoxy)ATP recycling system resulted in a significant increase in the conversion to all NTP products, furnishing 4 different NTPs in quantitative conversion. Natural (deoxy)NTPs were synthesized with 60 to >99% conversion and sugar- and base-modified NTPs were produced with 69 to >99% and 27 to 75% conversion, respectively. The presented method is suitable for the efficient synthesis of a wide range of natural and modified NTPs in a sustainable one-pot process.

The crystal structures of Thermus thermophilus CMP kinase complexed with a phosphoryl group acceptor and donor.

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open−closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.

Computational Overview of Mycobacterial Thymidine Monophosphate Kinase.

Tuberculosis (TB) ranks among the diseases with the highest morbidity rate with significantly high prevalence in developing countries. Globally, tuberculosis poses the most substantial burden of mortality. Further, a partially treated tuberculosis patient is worse than untreated; they may lead to standing out as a critical obstacle to global tuberculosis control. The emergence of multi-drug resistant (MDR) and extremely drug-resistant (XDR) strains, and co-infection of HIV further worsen the situation. The present review article discusses validated targets of the bacterial enzyme thymidine monophosphate kinase (TMPK). TMPKMTB enzyme belongs to the nucleoside monophosphate kinases (NMPKs) family. It is involved in phosphorylation of TMP to TDP, and TDP is phosphorylated to TTP. This review highlights structure elucidation of TMP enzymes and their inhibitors study on TMP scaffold, and it also discusses different techniques; including molecular docking, virtual screening, 3DPharmacophore, QSAR for finding anti-tubercular agents.

Identification of fish CMPK2 as an interferon stimulated gene against SVCV infection

Cytidine/uridine monophosphate kinase 2 (CMPK2) is known as a nucleoside monophosphate kinase in mitochondria to maintains intracellular UTP/CTP, and could be induced by immunostimulants LPS and Poly (I:C) in mammals, suggesting its potential antiviral and antibacterial role. In this study, CMPK2 was cloned and characterized in Fathead minnow (FHM) cells. In vivo analysis of tissue distribution revealed that CMPK2 transcript was detected in all the tissues of zebrafish (Danio rerio) examined in this study, particularly abundant in liver, spleen and kidney. In addition, indirect immunofluorescence showed that CMPK2 was localized in the cytoplasm of FHM cells. Expression of CMPK2 mRNA was significantly up-regulated following challenge with Spring viraemia of carp virus (SVCV), poly(I:C), or zebrafish IFN1 and IFN3 both in vitro and in vivo. Furthermore, overexpression and RNA interference of CMPK2 in SVCV-infected FHM cells showed significantly antiviral effect. In summary, this study for the first time shows the presence and distribution of CMPK2 in different tissues of zebrafish, but also demonstrates its antiviral potential against SVCV infection in vivo. These new findings could contribute to explain the molecular mechanism of the CMPK2 mediated antiviral function.

Cytidine monophosphate kinase is inhibited by the TGF-β signalling pathway through the upregulation of miR-130b-3p in human epithelial ovarian cancer

Cytidine monophosphate kinase (CMPK), a member of the nucleoside monophosphate kinase family, plays an important role in the biosynthesis of nucleoside metabolism, DNA repair and tumour development. In this study, we demonstrated for the first time that CMPK was overexpressed in human ovarian epithelial borderline and malignant tumours using tissue microarray. Knockdown of CMPK significantly inhibited epithelial ovarian cancer (EOC) cell proliferation, migration and invasion. Furthermore, CMPK-shRNA inhibited PCNA, MMP-2, MMP-9 and vimentin expression, increased E-cadherin expression and arrested cell cycle at the G2/M phase. Suppression of CMPK resulted in a decrease of EOC cell microtissue formation and colony formation in vitro. Overexpression of miR-130b-3p decreased CMPK expression, whereas anti-miR-130b-3p increased CMPK expression. Moreover, TGF-β1 inhibited the expression of CMPK, which was blocked in the presence of a TGF-β type I receptor, SB431542, and was abolished by the inhibitor of miR-130b-3p, indicating that CMPK is regulated by the TGF-β signalling pathway through the upregulation of miR-130b-3p. Thus, our data identify that overexpression of CMPK occurs in EOC and reveal a mechanism underlying the regulation of CMPK by the TGF-β signalling pathway. We could consider CMPK as an EOC biomarker and targeting CMPK by decreasing its expression may be beneficial in patients with EOC.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

AbstractStates along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Clinico-pathological correlation in adenylate kinase 5 autoimmune limbic encephalitis

Autoantibodies associated with autoimmune limbic encephalitis (ALE) have been well-characterized, with intracellular neuronal antibodies being less responsive to immunotherapy than antibodies to cell surface antigens. Adenylate kinase 5 (AK5) is a nucleoside monophosphate kinase vital for neuronal-specific metabolism and is located intracellularly in the cytosol and expressed exclusively in the brain. Antibodies to AK5 had been previously identified but were not known to be associated with human disease prior to the report of two patients with AK5-related ALE (Tuzun et al., 2007). We present the complete clinical picture for one of these patients and the first reported neuropathology for AK5 ALE.

Purification and characterization of guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi.

Guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi which is involved in reversible transfer of phosphate groups from ATP to GMP, was cloned, expressed and characterized. The native molecular mass of BmGK was found to be 45 kDa as determined by size exclusion chromatography and glutaraldehyde cross-linking which revealed that the protein is homodimer in nature. This is a unique characteristic among known eukaryotic GKs. GMP and ATP served as the most effective phosphate acceptor and donor, respectively. Recombinant BmGK utilized both GMP and dGMP, as substrates showing Km values of 30 and 38 μ m, respectively. Free Mg+2 (un-complexed to ATP) and GTP play a regulatory role in catalysis of BmGK. The enzyme showed higher catalytic efficiency as compared with human GK and showed ternary complex (BmGK-GMP-ATP) formation with sequential substrate binding. The secondary structure of BmGK consisted of 45% α-helices, 18% β-sheets as revealed by CD analysis. Homology modelling and docking with GMP revealed conserved substrate binding residues with slight differences. Differences in kinetic properties and oligomerization of BmGK compared with human GK can provide the way for design of parasite-specific inhibitors.

Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology.

Insight from the structural molecular model of cytidylate kinase from Mycobacterium tuberculosis

Mycobacterium tuberculosis is a gram-positive bacterium causes tuberculosis in human. H37Rv strain is a pathogenic strain utilized for tuberculosis research. The cytidylate mono-phosphate (CMP) kinase of Mycobacterium tuberculosis belongs to the family nucleoside mono-phosphate kinase (NMK), this enzyme is required for the bacterial growth. Therefore, it is important to study the structural and functional features of this enzyme in the control of the disease. Hence, we developed the structural molecular model of the CMP kinase protein from Mycobacterium tuberculosis by homology modeling using the software MODELLER (9v10). Based on sequence similarity with protein of known structure (template) of Mycobacterium smegmatis (PDB ID: 3R20) was chosen from protein databank (PDB) by using BLASTp. The energy of constructed models was minimized and the qualities of the models were evaluated by PROCHECK and VERRIFY-3D. Resulted Ramachandran plot analysis showed that conformations for 100.00% of amino acids residues are within the most favored regions. A possible homologous deep cleft active site was identified in the Model using CASTp program. Amino acid composition and polarity of that protein was observed by CLC-Protein Workbench tool. Expasy's Prot-param server and CYC_REC tool were used for physiochemical and functional characterization of the protein. Studied of secondary structure of that protein was carried out by computational program, ProFunc. The structure is finally submitted in Protein Model Database. The predicted model permits initial inferences about the unexplored 3D structure of the CMP kinase and may be promote in relational designing of molecules for structure-function studies.

Elucidation of Mg2+binding activity of adenylate kinase fromMycobacterium tuberculosisH37Rv using fluorescence studies

Adenylate kinase (AK) is a small ubiquitous enzyme that catalyzes the reversible transfer of the terminal phosphate group from adenine triphosphate (ATP): magnesium ion (Mg²⁺) to adenine monophosphate (AMP) to form two molecules of adenine diphosphate (ADP). AK thus maintains the homeostasis of adenine nucleotides in eukaryotes and prokaryotes. Because the [ATP]/[ADP] ratio is an important parameter in energy regulation in cells, Mg²⁺-activated AK has an important biological role, particularly in the case of bacteria, as imbalance in the ratio of [ATP]/[ADP] has been associated with alteration in its DNA supercoiling state. In the present study, magnesium-binding assays were carried out by systematically varying the concentrations of Mg²⁺, protein, AMP, ATP, and indicator in kinetic experiments. We report evidence that during magnesium-binding assay, the fluorescence level of the indicator "Mag-Indo-1" changes with protein concentration, suggesting that magnesium ions are binding to AK. The dual activity of AK both as nucleoside monophosphate and diphosphate kinases suggests that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism. According to the proposed model, the magnesium-activated AK exhibits an increase in its forward reaction rate compared with the inactivated form. These findings imply that Mg²⁺ could be an important regulator in the energy signaling network in cells.

Borononucleotides as Substrates/Binders for Human NMP Kinases: Enzymatic and Spectroscopic Evaluation

Borononucleotides are a family of natural nucleotide monophosphate analogues with a 5'-boronic acid function. As B-O-P linkages are known to be unstable in solution, we evaluated the ability of borononucleotides to be recognized by nucleoside monophosphate kinases and eventually foil the phosphorylation process. In this context, and with the idea of probing the influence of their size, shape, and flexibility, a library of borononucleotides were synthetized starting from the borononucleotide analogue of thymidine, which was shown to behave as a slow substrate of human TMP kinase. This study thus constitutes a good starting point for the development of new monophosphate mimics as potential substrates or ligands for NMP kinases.

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

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.

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation †

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(–) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

Transgenic cardiac-targeted overexpression of human thymidylate kinase

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the α-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In ‘2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3′-azido-3′deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.