Direct adenylation from 5'-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase.
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ABSTRACT:
SUBMITTER: Yang Z
PROVIDER: S-EPMC9303275 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The N-terminal kinase domain belongs to the P-loop phosphotransferase superfamily. The kinase is distinguished by a tunnel-like active site with separate entrances on opposite sides of the protein for the NTP phosphate donor and a 5'-OH single-stranded oligonucleotide phosphate acceptor. Here, we probed by mutagenesis the roles of individual amino acids that comprise the acceptor binding site. We thereby identified Glu57 as an important residue, by virtue of its participation in a salt bridge network with two catalytic residues identified previously: Arg38, which binds the 3'-phosphate of the terminal 5'-OH nucleotide, and the putative general base Asp35 that contacts the nucleophilic 5'-OH group. The 5'-OH nucleoside fits into a pocket lined by aliphatic amino acids (Val131, Pro132 and Val135) that make van der Waals contacts to the nucleobase. Whereas subtraction of these contacts by single alanine substitutions for Val131 or Val135 and glycine for Pro132 had modest effects on kinase activity, the introduction of bulkier phenylalanines for Val131 and Val135 were deleterious, especially V131F, which severely impeded both substrate binding (increasing K(m) by 15-fold) and catalysis (decreasing k(cat) by 300-fold).
| S-EPMC2831316 | biostudies-literature
Project description:The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development.
| S-EPMC3344967 | biostudies-literature
Project description:T4 phage polynucleotide kinase (PNK) displays 5'-hydroxyl kinase, 3'-phosphatase and 2',3'-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5' hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5' ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5'-GT DNA end than for a 5'-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.
| S-EPMC373337 | biostudies-literature
Project description:Ligases conduct the final stage of repair of DNA damage by sealing a single-stranded nick after excision of damaged nucleotides and reinsertion of correct nucleotides. Depending upon the circumstances and the success of the repair process, lesions may remain at the ligation site, either in the template or at the oligomer termini to be joined. Ligation experiments using bacteriophage T4 DNA ligase were carried out with purine lesions in four positions surrounding the nick site in a total of 96 different duplexes. The oxidized lesion 8-oxo-7,8-dihydroguanosine (OG) showed, as expected, that the enzyme is most sensitive to lesions on the 3' end of the nick compared to the 5' end and to lesions located in the intact template strand. In general, substrates containing the OG.A mismatch were more readily ligated than those with the OG.C mismatch. Ligations of duplexes containing the OA.T base pair (OA = 8-oxo-7,8-dihydroadenosine) that could adopt an anti-anti conformation proceeded with high efficiencies. An OI.A mismatch-containing duplex (OI = 8-oxo-7,8-dihydroinosine) behaved like OG.A. Due to its low reduction potential, OG is readily oxidized to secondary oxidation products, such as the guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides; these lesions also contain an oxo group at the original C8 position of the purine. Ligation of oligomers containing Gh and Sp occurred when opposite A and G, although the overall ligation efficiencies were much lower than those of most OG base pairs. Steady-state kinetic studies were carried out for representative examples of lesions in the template. Km increased by 90-100-fold for OG.C-, OI.C-, OI.A-, and OA.T-containing duplexes compared to that of a G.C-containing duplex. Substrates containing Gh.A, Gh.G, Sp.A, and Sp.G base pairs exhibited Km values 20-70-fold higher than that of the substrate containing a G.C base pair, while the Km value for OG.A was 5 times lower than that for G.C.
| S-EPMC2442820 | biostudies-literature
Project description: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. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A 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. The active site is composed of essential side chains that coordinate the beta phosphate of the NTP donor and the 3' phosphate of the 5' OH acceptor, plus a putative general acid that activates the 5' OH. The structure rationalizes the different specificities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.
| S-EPMC126130 | biostudies-literature
Project description:T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. 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. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. 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). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). 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.
| S-EPMC2761019 | biostudies-literature
Project description:The mycobacterial biotin protein ligase (MtBPL) globally regulates lipid metabolism in Mtb through the posttranslational biotinylation of acyl coenzyme A carboxylases involved in lipid biosynthesis that catalyze the first step in fatty acid biosynthesis and pyruvate coenzyme A carboxylase, a gluconeogenic enzyme vital for lipid catabolism. Here we describe the design, development, and evaluation of a rationally designed bisubstrate inhibitor of MtBPL. This inhibitor displays potent subnanomolar enzyme inhibition and antitubercular activity against multidrug resistant and extensively drug resistant Mtb strains. We show that the inhibitor decreases in vivo protein biotinylation of key enzymes involved in fatty acid biosynthesis and that the antibacterial activity is MtBPL dependent. Additionally, the gene encoding BPL was found to be essential in M. smegmatis. Finally, the X-ray cocrystal structure of inhibitor bound MtBPL was solved providing detailed insight for further structure-activity analysis. Collectively, these data suggest that MtBPL is a promising target for further antitubercular therapeutic development.
| S-EPMC3225891 | biostudies-literature
Project description:T4 polynucleotide kinase-phosphatase (Pnkp) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid-base catalyst Asp167. We report that Pnkp also has RNA 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2',3'-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3'-phosphoaspartyl)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + phosphoaspartyl-Pnkp → P(i) + Pnkp.
| S-EPMC3592404 | biostudies-literature
Project description:XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.
| S-EPMC2632933 | biostudies-literature
Project description:T4 polynucleotide kinase (Pnk) is a bifunctional 5'-kinase/3'-phosphatase that aids in the repair of broken termini in RNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate candidate catalytic residues within the 301 amino acid Pnk polypeptide. We found that alanine substitutions for Arg38 and Arg126 inactivated the 5'-kinase, but spared the 3'-phosphatase activity. Conservative substitutions of lysine or glutamine for Arg38 and Arg126 did not restore 5'-kinase activity. These results, together with previous mutational studies, highlight a constellation of five amino acids (Lys15, Ser16, Asp35, Arg38 and Arg126) that likely comprise the 5'-kinase active site. Four of these residues are conserved at the active sites of adenylate kinases (Adk), suggesting that Pnk and Adk are structurally and mechanistically related. We found that alanine substitutions for Asp165, Asp167, Arg176, Arg213, Asp254 and Asp278 inactivated the 3'-phosphatase, but spared the 5'-kinase. Conservative substitutions of asparagine or glutamate for Asp165, Asp167 and Asp254 did not revive the 3'-phosphatase activity, nor did lysine substitutions for Arg176 and Arg213. Glutamate in lieu of Asp278 partially restored activity, whereas asparagine had no salutary effect. Alanine substitutions for Arg246 and Arg279 partially inactivated the 3'-phosphatase; the conservative R246K change restored activity, whereas R279K had no benefit. The essential phosphatase residues Asp165 and Asp167 are located within a 165DxDxT169 motif that defines a superfamily of phosphotransferases. Our data suggest that the 3'-phosphatase active site incorporates multiple additional functional groups.
| S-EPMC100346 | biostudies-literature