Jones T.A., Zou,J.Y., Cowan,S.W. Hausmann S, Erdjument-Bromage H, Shuman S. Jacobson KB, Murphy JB, Das Sarma B. HHS Vulnerability Disclosure, Help We will keep you updated with any development. This exonuclease activity can proceed either in the 5'->3'direction, or in the 3'->5' direction. The digest was applied to a 4.5-ml column of Ni-agarose and the tag-free Pnkp protein was recovered in the flow-through fractions. cPhasing power = r.m.s. 2. A comparison of the crystal structure from the Stoddard group with a structurally characterized DxDxT protein established that the Pnkp phosphatase domain is a bona fide member of this superfamily (Galburt et al., 2002; Cho et al., 2001). Polynucleotide Kinase also catalyzes the removal of 3-phosphoryl groups from 3-phosphoryl polynucleotides, deoxynucleoside 3-monophosphates and deoxynucleoside 3-diphosphates (1). The primary structures of the kinase components of the T4 and eukaryotic Pnks are similar and four of the five amino acids that are essential for catalysis and comprise the active site in the T4 Pnk crystal structure (Lys15, Ser16, Asp35 and Arg126) are conserved in human and Schizosaccharomyces pombe Pnks (Jilani et al., 1999a; Karimi-Busheri et al., 1999; Meijer et al., 2002). official website and that any information you provide is encrypted We report the effects of 19 mutations at 12 conserved positions of Pnkp, which identify four residues (Asp187, Ser211, Lys258, and Asp277) as essential for activity. Careers. The two best matches were to the kinase domain of 6-phos phofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) [Protein Data Bank (PDB) code 1bif; z score 14.0; r.m.s.d. (2002), which reveals that Asp187 O1 and O2 form salt bridges to the Arg176 and Arg213 side chains, respectively. the contents by NLM or the National Institutes of Health. 3). and Deisenhofer,J. Please enable it to take advantage of the complete set of features! It has been proposed that the tetramer facilitates simultaneous processing of both termini of the broken tRNA substrate in vivo (Galburt et al., 2002), but there is no evidence that the two reactions are coupled during a single substrate binding event. 8600 Rockville Pike T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that play key roles in RNA and DNA repair. An aspartate is conserved at the equivalent position (Asp93) of CPT (Figure4). The https:// ensures that you are connecting to the Trp294, while making a hydrogen bond from N to Glu292 in the same subunit, engages in multiple cross-subunit van der Waals contacts to other hydrophobic side chains, including Val193, Leu196, and the Trp294 in the opposing subunit (Fig. We solved the structure by molecular replacement using the Stoddard groups structure as a search model. Bethesda, MD 20894, Web Policies Crystallographic dimer interfaces in Pnk and CPT. T4 Polynucleotide Kinase (PNK) is widely used to phosphorylate the 5' end of both DNA and RNA in labeling and ligation experiments. Eight of the mutants had activity similar to wild-type Pnkp: K198A (82%), G212A (37%), E219A (51%), T222A (94%), R229A (94%), E233A (45%), T251A (120%), and K253A (95%). We speculate that native Pnk homotetramer is assembled via high-affinity interactions between the C-terminal phosphatase domains (P) to form Pnk homodimers that are then converted into tetramers via lower-affinity interactions between the N-terminal kinase domains (K). and Lasko,D.D. The crystal structures were aligned by DALI. (2002). Aspartate was replaced by asparagine and glutamine, lysine by glutamine and arginine, and serine by threonine. and Li,H. Physical purity was evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. FOIA 2001 Jul 20;276(29):26868-74. doi: 10.1074/jbc.M103663200. The kinase monomer of the asymmetric unit makes contact with another monomer across a crystallographic 2-fold axis. and Kaufman,G. The Mg2+ of protomer B is depicted as a magenta sphere. Two of the essential phosphatase residues, Asp165 and Asp167, are located within a 165DxDxT169 motif that defines a superfamily of phosphotransferases that act through a covalent aspartyl phosphate intermediate (Collet et al., 1998; Burroughs et al., 2006). 5. We crystallized the N-terminal kinase domain Pnk(1181) and solved the structure by single isomorphous replacement with anomalous scattering using diffraction data from native and Hg-derivatized crystals. Direct adenylation from 5'-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase. Accessibility T4 Polynucleotide Kinase (PNK) is widely used to phosphorylate the 5 end of both DNA and RNA in labeling and ligation experiments. and Xaldecott,K.W. 2001 Jul 20;276(29):26868-74. doi: 10.1074/jbc.M103663200. Tete-Favier F, Cobessi D, Boschl-Muller S, Azza S, Branlant G, Aubry A. These data indicate that the C-terminal half of Pnk (amino acids 149301) comprises an autonomous 3-phosphatase domain with full catalytic activity. The .gov means its official. Structure-function analysis of the 3' phosphatase component of T4 polynucleotide kinase/phosphatase. 5). DNA kinase activity of T4 PNK is shown as a positive control. Although T4 Pnk is widely used to label DNA 5 ends in vitro, it is actually quite selective in phosphorylating DNAs with 5 single-strand extensions; T4 Pnk is poorly active or inactive on blunt duplex 5 OH termini or 5 OH termini that are recessed within duplex regions or at the junction of a 3 single-strand tail (Jilani et al., 1999b). Indeed, Arg176 is disordered in three of the protomers in tetramer 1 in our structure and is also disordered in the tetramer structures of Eastberg et al. These results suggest the existence of a domain boundary located between Arg126 and Asp165. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C . (1999) Automated MAD and MIR structure solution. 4B. These data establish a requirement for a carboxylate at positions 187 and 277 of T4 Pnkp and they indicate a tight steric constraint on the distance from the main-chain to the carboxylate at position 187, such that the active site is perturbed by the additional methylene group of glutamate. Lysozyme, PMSF, benzamidine, and Triton X-100 were added to final concentrations of 1 mg/ml, 0.2 mM, 1 mM and 0.2% respectively. Unable to load your collection due to an error, Unable to load your delegates due to an error, The amino acid sequence of T4 Pnkp from amino acids 159 to 301 is aligned with that of the phosphatase domain of the ORF86 polypeptide encoded by. Nat Commun. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. Lahiri SD, Zhang G, Dunaway-Mariano D, Allen KN. National Library of Medicine for 24h at 4C. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. The 5-kinase activity of Pnk(1147) was virtually identical to that of Pnk(1181) and glycerol gradient sedimentation indicated that Pnk(1147) was monomeric (data not shown). Because the phosphatase active site is composed of functional groups from a single protomer, with no apparent cross-subunit contributions (Fig. The inserts were sequenced completely to confirm the desired mutations and to exclude the acquisition of unwanted changes. SS is an American Cancer Society Research Professor. Polypeptides were visualized by staining with Coomassie Blue dye. Abele U. and Schulz,G.E. The protein sample (7mg/ml in 50mM TrisHCl pH7.5, 0.2M NaCl, 0.3M imidazole, 10% glycerol) was mixed with an equal volume of the reservoir buffer containing 100mM of sodium acetate pH4.6, 5mM DTT, 16% PEG-2000 monomethyl ether and 0.4M ammonium sulfate. Looking for a quick way to design experiments? The Pnkp dimer mutants retained kinase function, but were defective for 3 phosphatase activity. The reaction is reversible. 2013 Nov 15;425(22):4307-22. doi: 10.1016/j.jmb.2013.06.012. Product Source A E. coli strain that carries the cloned T4 Polynucleotide Kinase gene. Clin Transl Med. Careers, Unable to load your collection due to an error. Pairwise kinase-kinase and phosphatase-phosphatase dimer interfaces are located at the oblique and acute corners of the rhombus, respectively. The Mg2+ (cyan sphere) and sulfate ligands are shown. T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5-kinase and 3-phosphatase activities that function in nucleic acid repair. Indeed, mammalian Pnk requires that the phosphate acceptor have a minimum chain length of approximately eight deoxynucleotides for the kinase reaction to occur (Karimi-Busheri and Weinfeld, 1997). The Pnkp homotetramer was first crystallized by Galburt et al. Lillehaug J.R. (1977) Physicochemical properties of T4 polynucleotide kinase. Phosphatase activity was measured by the release of inorganic phosphate from deoxythymidine 3 monophosphate. Soltis D.A. Sedimentation analysis was performed as described under Methods. X-ray diffraction data for a native crystal were collected at the National Synchrotron Light Source (Brookhaven, NY) at beamline X9A using a 165-mm MAR CCD detector. The native size of the four phosphatase-defective mutant proteins was investigated by glycerol gradient sedimentation. Alternatively, primers for PCR can be ordered with 5' phosphate to avoid the need to separately phosphorylate the PCR product with a kinase. This site is protected by reCAPTCHA and the Google, Explore high-quality enzymes; now available as individual product. The other four Ala-cluster mutations elicited less drastic reduction in phosphatase activity, to between 18 and 27% of the wild-type level (Table IV). Wild-type Pnkp released 0.66 nmol of Pi from 3 dTMP per ng of protein in the linear range of enzyme-dependence, which translates into a turnover number of ~20 s1. Gaps in the alignment are indicated by dashes. A8.25-A8.26). The column was washed sequentially with 50 ml of buffer A (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol), 50 ml of 50 mM Imidazole in buffer A, and 50 ml of 100 mM imidazole in buffer A. The enzymatic phosphorylation of ribonucleic acid and deoxyribonucleic acid: further properties of the 5-hydroxyl polynucleotide kinase. Optically detected magnetic resonance study of the interactions of an arsenic(III) derivative of cacodylic acid with EcoRI methyl transferase. However, in the three other protomers of the homotetramer, Asp167 is oriented away from the phosphate-binding site and makes no contacts to either Ser211 or Lys258, leaving these functional groups available to bind the 3 phosphate substrate (Fig. A search of the protein structure database using DALI (Holm and Sander, 1993) found significant similarities between Pnk and other P-loop-containing enzymes. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. Asn190-Met192 and Met199-Tyr200 make van der Waals contacts at and across the dimer interface (Fig. We presume that the T4 Pnk reaction is promoted by contacts between the enzyme and the phosphate of ATP that serve to stabilize a pentacoordinate transition state. T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity. Curr Genet. The Pnk structure was solved by single isomorphous replacement with anomalous scattering. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. Terwilliger T.C. (1998) Crystal structures of. Alanine mutations were introduced into the full-length Pnk gene by using the two-stage PCR-based overlap extension method. Before Unable to load your collection due to an error, Unable to load your delegates due to an error. Arg287 and Gln295 are neighbors on the subunit interaction surface but they dont interact with each other. In contrast, the main chain of the loop that includes Asp167 (an essential residue and a putative general acid-base catalyst) varies in position from one protomer to the next, implying some conformational mobility. of 2.7 over 134C positions of equivalence to Pnk) (Hasemann et al., 1996; Izard and Ellis, 2000). and Kleppe,K. 24/7 automatic processing of online orders, Knowledgeable and professional Product & Technical Support. The kinase active site is composed of (i) a classical P-loop motif (9GxxGxGKS16) that coordinates the phosphate of the NTP donor; (ii) essential side chain Arg126, which also coordinates the NTP phosphate; (iii) essential side chain Arg38, which coordinates the 3 phosphate of the 5OH acceptor, plus (iv) essential side chain Asp35, a putative general acid that activates the 5OH for direct nucleophilic attack on the NTP phosphate. The assay time is 30 minutes and volume . bMFID (mean fractional isomorphous difference) = ||Fph| |Fp||/|Fp|, where Fp is the protein structure factor amplitude and |Fph| is the heavy-atom derivative structure factor amplitude. One module, composed of the -sheet and helices 1, 3, 4 and 5, forms a globular pedestal supporting the two sulfates (Figure2). The 3-phosphatase activity profile coincided with the sedimentation profile of the N148 polypeptide (data not shown). 5, see orange dashed lines), thereby comprising (with the lysine and serine side chains) the oxyanion hole of the Pnkp phosphatase. The T4 Polynucleotide Kinase is intended for molecular biology applications. sharing sensitive information, make sure youre on a federal The phosphatase specific activity of each protein was determined from the slope of the titration curve and then normalized to the specific activity of wild-type Pnkp (defined as 100%). 6. Si J, Zhou W, Fang Y, Zhou D, Gao Y, Yao Q, Shen X, Zhu C. Biosensors (Basel). The molecular contacts at the interface are predominantly hydrophobic, but also include hydrogen bonds between main chains (from the Asn31 amide to the Ile41 carbonyl), from side chain to main chain (Asn31 amide to Ser40 carbonyl) and from side chain to side chain side (Asp36 O1 to His44N; Asp36 O2 to His44N and Ser40 O). Protomer D has a disordered gap from aa 216-220 in another surface loop of the phosphatase domain. To test this hypothesis, we engineered a series of N-terminal Pnk truncation mutants, N138, N148 and N158, and produced the recombinant proteins in bacteria as His10-tagged fusions. Wang LK, Shuman S. Domain structure and mutational analysis of T4 polynucleotide kinase. To probe the contributions of quaternary structure to the phosphatase function of Pnkp, we introduced six double-alanine cluster mutations at amino acids located at or near the phosphatase dimer interface of the Pnkp crystal structure. T4 polynucleotide kinase/phosphatase crystallographic data. The distributions of Pnkp Ala-Ala mutants and the marker proteins catalase, BSA, and cytochrome. Nucleic Acids Res. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). The physiological substrates of T4 Pnk in vivo are broken tRNAs incised 5 to the wobble base of the anticodon loop (Amitsur et al., 1987). Model Contents: 12 Pnkp monomers containing 3542 protein residues. 4A). The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer. 4A and B) is consistent with previous findings that D278A and D278N mutations abolished 3 phosphatase activity, which was restored partially by a conservative D278E change (Wang and Shuman, 2002). official website and that any information you provide is encrypted Epub 2014 Jul 21. These results suggest that homodimerization of the phosphatase domain is important for phosphatase activity. and transmitted securely. The simplest explanation for the change in Pnkp quaternary structure is that the cluster mutations disrupted the phosphatase-phosphatase interface, while sparing the kinase-kinase interface. Marker proteins bovine serum albumin (BSA; 66 kDa), ovalbumin (45 kDa) and cytochrome c (13 kDa) were included as internal standards. T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. Incubation for 4 hours at 37C resulted in less than 1.0% release of TCA-soluble counts. and Uhlenbeck,O.C. and transmitted securely. The 2.0 crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5 OH acceptor side that can accommodate a single-stranded polynucleotide. (1988) Refined structure of porcine cytosolic adenylate kinase at 2.1 resolution. Aside from the flexible loops, each protomer shows an average root mean square deviation of ~0.5 for 280 C atoms. This covalent modification of cysteine by arsenic is well described and depends on the presence of both cacodylate and DTT (Jacobson et al., 1972; Tsao and Maki, 1991; Maignan et al., 1998). 7). (2001) XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair. and Lapthorn,A.J. The C-terminal half of the Pnk protomer, comprising an autonomous phosphatase catalytic domain (P in Figure7), forms a stable homodimer in solution at low protein concentrations in the absence of its substrate. T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. Fig. The Asp167 carboxylate forms an ion pair with Arg213 in every protomer; Arg213 is strictly essential for 3 phosphatase activity (Wang and Shuman, 2002). The rationale for the homodimeric quaternary structure of T4 Pnkp is not clear. 2014 Jan 31;289(5):3094-103. doi: 10.1074/jbc.M113.536482. Panet A., van de Sande,J.H., Loewen,P.C., Khorana,H.G., Raae,A.J., Lillehaug,J.R. The network of sulfate to P-loop interactions in the Pnk crystal typifies contacts made to the nucleotide phosphate in the structures of numerous other P-loop-containing phosphotransferases; therefore, we surmise that this sulfate demarcates the NTP binding site of Pnk. Fig. and Shuman,S. We surmise that dimerization of the C-terminal domain of Pnkp is important for phosphatase function. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected. Two sulfate ions bound to Pnk, one to the P-loop and a second to 2 and the loop connecting 3 to 4, demarcate the NTP and phosphate-acceptor binding sites, respectively (see below). Three of the six magnesium coordination sites entail direct contacts to protein side-chain or main-chain oxygens; two sites are occupied by waters coordinated by protein side chains, and the sixth site entails metal coordination to a phosphate oxygen. government site. and Phillips,G.N. Large-Scale Molecular Evolutionary Analysis Uncovers a Variety of Polynucleotide Kinase Clp1 Family Proteins in the Three Domains of Life. T4 PNK catalyzes the transfer and exchange of the terminal gamma position phosphate of ATP to the 5-hydroxyl terminus of double- and single-stranded DNA, RNA and nucleoside 3-monophosphate molecules. 2022 Jul;12(7):e987. The second sulfate is coordinated to the main-chain amide nitrogen and the side-chain O of Thr86 (in the 34 loop) and to both terminal guanidinium nitrogens of Arg38 (in 2) (Figure6). Hydrogen-bonding and ionic interactions of these side chains are indicated by dashed lines. 2002 Feb 15;30(4):1073-80. doi: 10.1093/nar/30.4.1073. 1). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. (1998) The three-dimensional structure of shikimate kinase. Refinement Residuals: 20.0 3.0 (3.11 3.0 ), I I: 0.0126 / 1.503 (Engh and Huber parameters). The specific activities of the 12 Ala-mutants were determined by enzyme titration and normalized to that of the wild-type Pnkp (Table I). CPT is a dimer under physiological solution conditions; Izard and Ellis (2000) proposed that the CPT subunit interface that superimposes on Pnk represents the physiological homodimerization surface. 7. The native sizes of the six Ala-cluster mutants were gauged by glycerol gradient sedimentation with internal standards (Fig. The Mg2+ of Scp1 is depicted as a cyan sphere and the associated waters as red spheres. To further evaluate the contributions of Asp187, Ser211, Lys258 and Asp277 to the 3 phosphatase reaction, we tested the effects of conservative substitutions. Construction of linker-scanning mutations by oligonucleotide ligation. 2019 Oct 1;11(10):2713-2726. doi: 10.1093/gbe/evz195. Wang L.K. Elkins JM, Soundararajan M, Yang X, Papagrigoriou E, Sundstrom M, Edwards A, Arrowsmith C, Weigelt J, Doyle DA. Purity was assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the protein of interest band in the diluted sample. 1). 100u. A Coomassie Blue-stained gel is shown. 2013 Jan 7;41(1):355-65. doi: 10.1093/nar/gks977. The dimers were superimposed by a least-squares fit in programO and the images were offset vertically in SETOR. In addition, the Pnk crystal structure shows that Lys129 and Arg47 project downward from the lid toward the active site (Figure6). The site is secure. The six full-length Pnkp-Ala/Ala mutants were produced in bacteria as His10-tagged fusions and purified from soluble bacterial extracts by Ni-agarose chromatography in parallel with wild-type Pnkp; SDS-PAGE analysis confirmed that the preparations were of equivalent purity (not shown). Pnkp crystallized in space group P21212 (a=124.63 , b= 128.05 , c=357.12 ; ,, = 90). Structure of a tRNA repair enzyme and molecular biology workhorse: T4 polynucleotide kinase. Please enable it to take advantage of the complete set of features! T4 PNK catalyzes the transfer and exchange of the terminal gamma position phosphate of ATP to the 5-hydroxyl terminus of double- and single-stranded DNA, RNA and nucleoside 3-monophosphate molecules. The enzyme may be used to phosphorylate RNA, DNA and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning. Phosphatase activity was measured by the release of inorganic phosphate from deoxythymidine 3 monophosphate (Becker and Hurwitz, 1967; Cameron and Uhlenbeck, 1977). This phosphate transfer is commonly referred to as a kinase or phosphorylation reaction ([gamma-32 P]ATP is often used in the reaction).Nucleic acids with a 5'-hydroxyl (OH) group can be added directly to a kinase reaction. Recombinant His-Pnk(1181) was purified from soluble E.coli extracts by Niagarose chromatography as described previously (Wang and Shuman, 2001), except that Triton X-100 was omitted from the lysis and column buffers. This closed conformation is incompatible with catalysis. The sulfate ion near the phosphatase active site is situated between Arg279 and Arg246 (Figs. Both interaction surfaces involve contributions from the equivalent secondary structure elements, particularly 2 and 3 and their adjacent loops (Figure5). The Pnk(1181) protein sedimented as a monomer in a glycerol gradient (Wang and Shuman, 2001). The biochemical and crystallographic results presented here engender a model for how the 5-kinase and 3-phosphatase enzymatic domains might be arranged in the native T4 Pnk homotetramer (Figure7). The direction of sedimentation is from the right (top of gradient) to the left (bottom). The crystal structure of the T4 kinase domain indicates that the tunnel aperture leading to the 3 phosphate binding site of the phosphate acceptor (Figure3) is too narrow to allow facile ingress of duplex nucleic acid, but can readily accommodate a single-stranded polynucleotide. 5 shows a superposition of the open active site of the Pnkp D protomer (green carbon atoms) on the active site of human Scp1, a DxD-family protein serine phosphatase that dephosphorylates the carboxyl-terminal domain of RNA polymerase II (Kamenski et al., 2004). Karimi-Busheri F. and Weinfeld,M. Therefore, we infer that the contacts between Pnk and the second sulfate ion mimic its interactions with the 3 PO4 of the terminal nucleotide of the acceptor substrate. Bookshelf An official website of the United States government. Contacts are denoted by dashed lines. 3-Phosphatase activity in T4 polynucleotide kinase. The https:// ensures that you are connecting to the Both arginines engage in ionic interactions with the Asp254 carboxylate, which is itself essential for 3-phosphatase activity (Wang and Shuman, 2002). Accessibility Two enzyme-bound sulfates coordinated at the active site tunnel are shown as a stick models. The essential Lys15 and Arg126 side chains are poised for such a role. Reconstitution and structure of a bacterial Pnkp1-Rnl-Hen1 RNA repair complex. Because it was not possible to differentiate between these two products at 3.0 resolution, we modeled only a single non-bonded arsenic atom for this adduct. official website and that any information you provide is encrypted Bethesda, MD 20894, Web Policies 2023 Apr;64(4):100352. doi: 10.1016/j.jlr.2023.100352. The 2.0 A crystal structure . Otwinowski Z. and Minor,W. To further delineate the phosphatase active site and catalytic mechanism of Pnkp, we conducted two lines of investigation: (i) additional mutational analysis of the C-terminal phosphatase domain, guided by structure and sequence comparisons to a eukaryotic viral homolog (Galburt et al., 2002; Martins and Shuman, 2004); and (ii) crystallization of the native Pnkp homotetramer in the presence of sulfate, a phosphate mimetic. Epub 2017 Sep 19. Amino acids that line the homodimer interface are shown in stick representation; residues from the gray promoter are labeled in, Sedimentation analysis was performed as described under Methods. Images of the Pnk structure were prepared with SETOR (Evans, 1993). of 2.5 over 140C positions of equivalence to Pnk] and to chloramphenicol phosphotransferase (CPT) (PDB code 1qhs; z score 12.2; r.m.s.d. The kinase contains a consensus Walker A-box motif (9GxxGxGKS16) located between the first -strand and the first -helix. A convenient tool to build experimental workflows and find products to match your needs. We draw similar inferences for the Pnk kinase domain. Galburt EA, Pelletier J, Wilson G, Stoddard BL. Crystals were grown over 2days to 1week. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. 7. The distributions of Pnkp (either WT, D187A, S211A, K258A or D277A as indicated) and the marker proteins catalase, BSA, and cytochrome, (A) Stereo view of the superimposed phosphatase active sites from Pnkp protomers A, B, C, and D (with the carbon atoms colored cyan for chain A, beige for chain B, yellow for chain C, and magenta for chain D) and the active site from the symmetrical Pnkp homotetramer structure reported previously (with carbon atoms in green). Bookshelf See this image and copyright information in PMC. Cho H, Wang W, Kim R, Yokota H, Damo S, Kin SH, Wemmer D, Kustu S, Yan D. BeF, Collet JF, Stroobant V, Pirard M, Delpierre G, Van Schaftingen E. A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal D. Eastberg JH, Pelletier J, Stoddard BL. 7). Incubation for 4 hours at 37C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis. The enzyme is used to remove 3-phosphoryl groups from nucleotide/nucleoside or DNA and RNA in studies of gene expression and gene function. The enzyme is supplied with a 10X PNK Buffer (B9040) containing 700 mM Tris-HCl, 100 mM MgCl2, 50 mM DTT; pH 7.6 at 25C. It also can catalyze the exchange of 5-terminal phosphate groups. One or more residues or side chains within this loop were disordered and therefore omitted from the final model in each of the protomers. INTRODUCTION. J Biol Chem. The delivery time takes two to three weeks at the moment. The Asp254 backbone amide also donates a hydrogen bond to the sulfate. The crystal structure of the isolated kinase domain (aa 1-153) revealed that the kinase is also a homodimer (Wang et al., 2002). Myllykoski M, Raasakka A, Lehtimki M, Han H, Kursula I, Kursula P. J Mol Biol. Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021 and. Data were integrated, scaled and merged using DENZO and SCALEPACK (Otwinowski and Minor, 1997). Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. official website and that any information you provide is encrypted Arg126 of Pnk is situated within a motif, 122RxxxR126, that is conserved in the structures of CPT and Adk enzymes (Figure4). In sum, the Pnkp structures now suggest plausible roles (in catalysis, substrate binding, or active site organization) for ten of the twelve key residues identified by mutagenesis: Asp165, Asp167, Ser211, Arg213, Arg246, Asp254, Lys258, Asp277, Asp278 and Arg279. The kinase can be divided into two sub-domains. government site. The Adk reaction is superficially similar to that of Pnk insofar as it entails nucleophilic attack on the phosphorus of ATP and is dependent on a divalent cation cofactor. Aliquots (40 g) of the Pnkp preparations were mixed with catalase (30 g), BSA (30 g), and cytochrome c (30 g) and the mixtures were applied to 4.8-ml 1530% glycerol gradients containing 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA, 2.5 mM DTT, 0.1% Triton X-100. As a library, NLM provides access to scientific literature. 1). Novogrodsky A, Hurwitz J. T4 Polynucleotide Kinase can be used to 5' end label DNA and RNA because it catalyzes the transfer and exchange of phosphate from ATP to the 5 -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3-monophosphates.Polynucleotide Kinase also catalyzes the removal of 3-phosphoryl groups from 3-phosphoryl polynucleotides, deoxynucleoside 3 . (|Fh|/E, where |Fh| is the heavy-atom structure factor amplitude and E is the residual lack of closure error). The high ionic strength of the kinase domain crystallization buffer and the occupancy of both substrate sites by sulfate may have stabilized a homodimer of the isolated kinase domain in the crystal, whereas the interactions of the N-terminal domain in standard purification buffers would require prior dimerization of the phosphatase domains. (1973) Physical characterization and simultaneous purification of bacteriophage T4 induced polynucleotide kinase, polynucleotide ligase, and deoxyribonucleic acid polymerase. Higher order similarities between Pnk and CPT are revealed by the conservation of a predominantly hydrophobic dimer interface across their respective crystallographic 2-fold axes (Figure5). The imputed role of Asp278 carboxylate in metal binding (Figs. T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the -phosphate from ATP to the 5-terminus of polynucleotides or to mononucleotides bearing a 3-phosphate group. During T4 infection, Pnkp participates in an RNA-based immune response whereby the bacterium attempts to thwart T4 protein . An official website of the United States government. (2004). Polynucleotide kinase (Pnk) was discovered by the Richardson and Hurwitz laboratories in T4 and T2 bacteriophage-infected Escherichia coli (Richardson, 1965; Novogrodsky and Hurwitz, 1966; Novogrodsky et al., 1966). and Murray,N.E. The tunnel opening on the opposite face of the protein, extending from the 3-phosphate site (sulfate2) out to the surface, is lined with basic side chains (Arg92, Lys51 and Lys54) and affords a plausible path for ingress of the 5 end of a polynucleotide to the T4 kinase active site. 47.00. Proc Natl Acad Sci U S A. Two sulfate ions (derived from the ammonium sulfate included in the crystallization buffer) were bound by the kinase active site in positions corresponding to the phosphate of the NTP donor and the phosphate of the 5-terminal HONp-N dinucleotide of the polynucleotide phosphate acceptor (not shown). A Trojan horse transition state analogue generated by MgF. 1; unimportant residues are denoted by +). The Scp1 structure (depicted with beige carbon atoms) captures the stable covalent apspartyl-BeF3 adduct as a mimetic of the labile aspartyl-phosphate intermediate. The .gov means its official. (1965) Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected. The phosphatase homodimer interface, A stereo view is shown with one phosphatase protomer, Fig. Recombinant protein production was induced by adjusting exponentially growing cultures (100 ml) to 0.3 mM IPTG and incubating them at 17C for 15 h with continuous shaking. National Library of Medicine 4A depicts the Mg2+ and sulfate ligands from protomer B. Whitehouse C.J., Taylor,R.M., Thistlethwaite,A., Zhang,H.M., Karimi-Busheri,F., Lasko,D.D., Weinfeld,M. Rfree = R based on 5% of the data withheld for the cross-validation test. 6). Reactions mixtures (10 l) containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 5 mM DTT, 25 M [32P]ATP, 1 mM 3 CMP (Sigma) and 0, 4.7, 9.4, 18.8, 37.5, or 75 ng of wild-type or mutant Pnkp were incubated for 20 min at 37C. Velocity sedimentation of phosphatase-defective Pnkp-Ala, Fig. PubMed Abstract: T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. The specific activity of the K129A and R47A mutants in phosphorylating 3 CMP were 51 and 77% of the activity of wild-type Pnk, respectively (data not shown). Incorporates labeled phosphates at the 5 end of nucleic acids, Catalyzes transfer and exchange of phosphate to the 5-OH of ssDNA and RNA, Catalyzes transfer and exchange of phosphate to the 5-OH of dsDNA, Removes 3 phosphoryl groups from DNA and RNA or a nucleotide/nucleoside, labeling the 5 ends of DNA or RNA for use, phosphorylating oligodeoxynucleotide linkers or other DNA molecules prior to ligation, end-labeling oligodeoxynucleotide primers for use in sequencing reactions, dephosphorylating the 3-ends of RNA in the absence of ATP, in an exchange reaction to label the 5 ends of DNA or RNA molecules that have unlabeled 5 phosphates. The sulfate mimicking the phosphate is also engaged in a bidentate interaction with the terminal guanidinium nitrogens of Arg126. In light of the above results defining the proximal boundary of the phosphatase domain near position 149, we constructed a shorter N-terminal derivative, Pnk(1147), and purified the His-tagged recombinant protein from bacteria. 4A and B). Federal government websites often end in .gov or .mil. Although the C position of Arg213 does not change, the arginine adopts different rotamer conformations to accommodate the subtle backbone movements and side chain rotations of Asp167. Protein concentrations were determined by using the BioRad dye reagent with bovine serum albumin as the standard. 2014 Aug 5;111(31):11317-22. doi: 10.1073/pnas.1409203111. Reaction of cacodylate with organic thiols. Abelson J., Trotta,C.R. The P-loop main-chain amides and side chains Lys15, Ser16 and Arg126 coordinate a sulfate corresponding to the phosphate of the NTP substrate. Martins A, Shuman S. Characterization of a baculovirus enzyme with RNA ligase, polynucleotide 5 kinase and polynucleotide 3 phosphatase activities. N-terminal deletion mutants Pnk(139301) [N138], Pnk(149301) [N148] and Pnk(159301) [N158] were constructed by PCR amplification with mutagenic sense-strand primers that introduced an NdeI restriction site and a methionine codon in lieu of the codon for 138, 148 or 158. T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5-kinase and 3-phosphatase activities that function in nucleic acid repair. A model of T4 Pnk quaternary structure. (1967) The enzymatic cleavage of phosphate termini from polynucleotides. We showed previously that the N-terminal segment of Pnk from amino acids 1181 (corresponding to a chymotrypsin-resistant fragment characterized by limited proteolysis) retained 5-kinase activity, albeit at an order of magnitude lower specific activity than full-length Pnk. 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