Molecular interactions involving Escherichia coli nucleoside diphosphate kinase

Nucleoside diphosphate kinase plays a distinctive metabolic role as the enzyme poised between the last reaction of deoxyribonucleoside triphosphate (dNTP) biosynthesis and the DNA polymerization apparatus. In bacteriophage T4 infection, NDP kinase is one of very few enzymes of host cell origin to participate in either dNTP synthesis or DNA replication. Yet NDP kinase forms specific contacts with phage-coded proteins of dNTP and DNA synthesis. This article summarizes work from our laboratory that identifies and characterizes these interactions. Despite these specific interactions, the enzyme appears to be dispensable, both for T4 replication and for growth of the host, Escherichia coli, because site-specific disruption of ndk, the structural gene for NDP kinase, does not interfere with growth of the host cell and only partly inhibits phage replication. However, ndk disruption unbalances the dNTP pools and stimulates mutagenesis. We discuss our attempts to understand the basis for this enhanced mutagenesis.     

Purification and characterization of the deoxynucleoside monophosphate kinase of bacteriophage T5

Deoxynucleoside monophosphate kinase (dNMP kinase) of bacteriophage T5 (EC 2.7.4.13) was purified to apparent homogeneity from phage-infected Escherichia coli cells. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gel showed that the enzyme has a molecular mass of about 29 kDa. The molecular mass of dNMP kinase estimated by analytical equilibrium ultracentrifugation turned out to be 29.14 +/- 3.03 kDa. These data suggest that the enzyme exists in solution as a monomer. The isoelectric point of dNMP kinase was found to be 4.2. The N-terminal amino acid sequence, comprising 21 amino acids, was determined to be VLVGLHGEAGSGKDGVAKLII. A comparison of this amino acid sequence and those of known enzymes with a similar function suggests the presence of a nucleotide-binding site in the sequenced region.     

Isolation and Partial Characterization of Bacteriophage T5 Mutants Deficient in the Ability to Induce Deoxynucleoside Monophosphate Kinase

Two mutants of bacteriophage T5 deficient in the ability to induce wild-type levels of deoxynucleoside monophosphate kinase were isolated and partially characterized. Both mutations were demonstrated to be in a structural gene for the kinase. One of the mutants, designated dnk 10, induces no detectable levels of dCMP, dGMP, or dTMP kinase activity. Because the mutant can successfully infect nonpermissive cells, phage-induced deoxynucleoside monophosphate kinase appears to be an unessential function for phage production. DNA synthesis in dnk 10-infected cells, however, is reduced to 30% of that observed in wild-type-infected cells; phage production is reduced by a comparable amount. The dnk mutation has been mapped and located on the "C" region of the T5 genetic map, 6.3 map units from the C1 locus.     

Investigation of the Substrate Recognition of Drosophila melanogaster Nucleoside Kinase by Site Directed Mutagenesis

The deoxyribonucleoside kinase of Drosophila melanogaster (Dm‐dNK) has a broad substrate specificity and a higher catalytic rate than other known deoxyribonucleoside kinases. Therefore it is a natural candidate for possible use as a suicide gene in combined gene/chemotherapy of cancer. We have performed site directed mutagenesis and tested different truncated forms of the enzyme in order to increase the affinity for ganciclovir.     

A DNA polymerase from Ustilago maydis. 2. Properties of the associated deoxyribonuclease activity.

The polymerase and deoxyribonuclease activities of the purified Ustilago maydis DNA polymerase coeluted from a hydroxyapatite column, cosedimented in sucrose gradients in both the absence and presence of salt, possessed similar thermolabilities and reaction requirements. These observations suggest that both activities are associated with the same enzyme and that the deoxyribonuclease activity is not a contaminant. The initial rate of degradation of native 3'-end-group-labelled DNA was similar to that of a heat-denatured substrate, but the final extent was greater for the former. The enzyme exhibits a high specificity for degradation of DNA in a 3' leads to 5' direction. The degradation of a DNA template was inhibited by the presence of the deoxyribonucleoside triphosphates necessary for simultaneous DNA synthesis, but not that of the newly synthesised DNA. About 50%, 29% and 13% of the purine, cytosine and thymine deoxyribonucleotide residues incorporated by the enzyme into DNA respectively, were subsequently excised when monitored by the resulting conversion of the triphosphate substrates to free monophosphate. The majority of the purine deoxyribonucleoside monophosphates appear after the synthetic phase of the reaction has ceased. In many respects, therefore, the deoxyribonuclease activity of the U. maydis DNA polymerase is similar to the bacteriophage T4-induced enzyme.     

Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens

To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.     

The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

The amino acid residues essential for the enzymatic activity of bacteriophage T5 deoxyribonucleoside monophosphate kinase were determined using a computer model of the enzyme active site. By site-directed mutagenesis, cloning, and gene expression in E. coli, a series of proteins were obtained with single substitutions of the conserved active site amino acid residues—S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, and E176Q. After purification by ion exchange and affine chromatography electrophoretically homogeneous preparations were obtained. The study of the enzymatic activity with natural acceptors of the phosphoryl group (dAMP, dCMP, dGMP, and dTMP) demonstrated that the substitutions of charged amino acid residues of the NMP binding domain (R130, R172, D170, and E176) caused nearly complete loss of enzymatic properties. It was found that the presence of the OH-group at position 17 was also important for the catalytic activity. On the basis of the analysis of specific activity variations we assumed that arginine residues at positions 130 and 172 were involved in the binding to the donor γ-phosphoryl and acceptor α-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. Disproportional changes in enzymatic activities of partially active mutants, G137A, T138A, T17N, Q134A, S13A, and D16N, toward different substrates may indicate that different amino acid residues participate in the binding to various substrates.     

Mosquito has a single multisubstrate deoxyribonucleoside kinase characterized by unique substrate specificity

In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine deoxyribonucleosides than the fruit fly multisubstrate deoxyribonucleoside kinase (EC 2.7.1.145). In addition, Ag-dNK could also phosphorylate some medically interesting nucleoside analogs, like stavudine (D4T), 2-chloro-deoxyadenosine (CdA) and 5-bromo-vinyl-deoxyuridine (BVDU). Although the biological significance of multisubstrate deoxyribonucleoside kinases and their diversity among insects remains unclear, the observed variation provides a whole range of applications, as species specific and highly selective targets for insecticides, they have a potential to be used in the enzymatic production of various (di-)(deoxy-)ribonucleoside monophosphates, and as suicide genes in gene therapy.     

A Cre/loxP-mediated self-activating gene excision system to produce marker gene free transgenic soybean plants

Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either β-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

A novel nucleotide kinase encoded by gene 1.7 of bacteriophage T7

Gene 1.7 of bacteriophage T7 confers sensitivity of both phage T7 and its host Escherichia coli to dideoxythymidine (ddT). We have purified the product of gene 1.7, gp1.7. It exists in two forms of molecular weight 22 181 and 17 782. Only the C‐terminal half of the protein is required to confer ddT sensitivity. We show that gp1.7 catalyses the phosphorylation of dGMP and dTMP to dGDP and dTDP, respectively, by using either GTP, dGTP or dTTP as the phosphate donor. Either form of gp1.7 exhibit identical kinase activity as compared with wild‐type gp1.7 that contains a mixture of both forms. The K_m of 70 µM and Kcat of 4.3 s^?1 for dTMP are similar to those found for E. coli thymidylate kinase. However, unlike the host enzyme, gp1.7 efficiently catalyses the conversion of the chain‐terminating dideoxythymidylate (ddTMP) to ddTDP. This finding explains the sensitivity of phage T7 but not E. coli to exogenous ddT. Gp1.7 is unusual in that it has no sequence homology to any known nucleotide kinase, it has no identifiable nucleotide‐binding motif and its activity is independent of added metal ions. When coupled with nucleoside diphosphate kinase, gp1.7 exponentially converts dTMP to dTTP.     

Cloning and characterization of an adenosine kinase from Physcomitrella involved in cytokinin metabolism

Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G. was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain. The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa. Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome. The deduced amino acid sequence had a 52% identity with the human adenosine kinase. The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E. coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue. Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella.     

Localization of the dihydrofolate reductase gene on the physical map of bacteriophage T5

Summary Large quantities of dihydrofolate reductase are synthesized in bacteriophage T5 infected E. coli cells. Some evidence that this enzyme is the product of a viral gene was published by Mathews (1967). Further evidence is presented now by showing that the newly synthesized enzyme differs from the preexisting E. coli reductase in molecular weight and salt solubility.The expression of the T5 dihydrofolate reductase gene was not affected by deletions in the del region of the phage genome. The map position of the reductase gene was determined by marker rescue experiments designed as helped transfection procedure: When E. coli B cells were preinfected with T5 dihydrofolate reductase amber mutants, made competent, and transfected with T5 wild type DNA, viable phages were obtained. Wild type recombinant phages were observed, when the transfecting DNA had been digested with the restriction endonucleases EcoRI, HpaI, PstI, and SalI. No rescue occurred when the DNA had been digested with AluI, EcoRII, HindII, HindIII, MboII, Sau3A, and XbaI. Single EcoRI, HpaI, and SalI restriction fragments were isolated and found to rescue the dihydrofolate reductase gene. Their common overlapping sequence corresponds to 8.6% of the phage DNA, a segment of about 10,000 base pairs length, which extends from position 0.37 to position 0.46 of the physical map. After cleaving this segment at its single HindIII recognition site marker rescue no longer occurred. From these results it was concluded that the dihydrofolate reductase gene either lies at or very close to this site at position 0.4.The helped transfection method was also used to rescue T5 mutants with defects in the genes C2 and D9. Gene C2 was localized on an EcoRI fragment that covers the DNA from map position 0.08 to map position 0.25. By localizing the two genes B3 and C2 on the restriction map of the T5 DNA a correlation of the genetic and the physical maps of the T5 genome has been established. Abbreviations. The symbols for T5 phages follow those of McCorquodale (1975) and the nomenclature for restriction nucleases that of Smith and Nathans (1973). kb=kilo base pairs     

Substrate specificity of T5 bacteriophage deoxyribonucleoside monophosphate kinase and its application for the synthesis of [α-<Superscript>32</Superscript>P]d/rNTP

Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d₂CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, k _m and k _cat were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [α-³²P]rNTP and [α-³²P]dNTP.     

A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to “shuttle” phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.     

Phage abortive infection of Bacillus licheniformis ATCC 9800; identification of the abiBL11 gene and localisation and sequencing of its promoter region

The virulent bacteriophage BL11 infects almost all Bacillus licheniformis strains tested, including the industrial bacitracin-producing B. licheniformis 19. B. licheniformis ATCC 9800, however, was virtually insensitive to phage BL11 infection, and all of the few surviving progeny phages proved to be mutants. The phage-resistance mechanism was neither inhibition of adsorption, nor restriction or exclusion provided by a resident prophage, but was, instead, of another type. Phage BL11 adsorbed well on to ATCC 9800 cells, its DNA was injected, but replication of phage DNA was inhibited and the infected cells died. Thus, the mechanism of phage resistance was identified as abortive infection (AbiBL11). The so-called abiBL11 gene was identified on the chromosome of strain ATCC 9800 by Tn917PF1 transposon mutagenesis. Part of the abiBL11 gene from the phage-sensitive ATCC 9800::Tn917PFI was cloned. Gene-disruption analysis, based on Campbell-type integration, showed that a 0.3-kb EcoRI fragment contained the 5′ end of abiBL11. The promoter region of abiBL11 was identified using promoter- and terminator-probe plasmids. The deduced sequence (206 amino acids) of the N-terminal part of abiBL11 showed no significant homology to known abortive-infection genes, but did show homology to a Saccharomycescerevisiae gene coding for a serine/threonine protein kinase (RCK1). Received: 1 April 1999 / Received revision: 12 July 1999 / Accepted: 13 July 1999