The stereochemical course of phosphoryl transfer catalysed by polynucleotide kinase (bacteriophage-T4-infected <i>Escherichia coli</i> B)

2012

Nucleic Acids Research

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 RNAOH, 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 RNAOH. Our results suggest that healing of 2′,3′-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3′-phosphoaspartyl)-Pnkp → RNA3′p + Pnkp → RNAOH + phosphoaspartyl-Pnkp → Pi + Pnkp.

Section: Introductionmentioning

confidence: 99%

Mechanism of RNA 2′,3′-cyclic phosphate end healing by T4 polynucleotide kinase–phosphatase

Das

2012

Nucleic Acids Research

“…Although it was initially proposed that T4 Pnk acts via a ping-pong mechanism with a phosphoenzyme intermediate (4), kinetic analysis favors a sequential mechanism in which both ATP and the 5Ј-OH polynucleotide bind the enzyme prior to dissociation of either product (5). The stereochemical course of the reaction entails inversion of configuration of the transferred phosphate, indicative of an in-line mechanism in which the polynucleotide 5Ј-OH directly attacks the ␥ phosphorus of ATP (6).…”

mentioning

confidence: 99%

Domain Structure and Mutational Analysis of T4 Polynucleotide Kinase

Wang

Journal of Biological Chemistry

2001

128

T4 polynucleotide kinase (Pnk) is the founding member of a family of 5-kinase/3-phosphatase enzymes that heal broken termini in RNA or DNA by converting 3-PO 4 /5-OH ends into 3-OH/5-PO 4 ends, which are then suitable for sealing by RNA or DNA ligases. Here we employed site-directed mutagenesis and biochemical methods to dissect the domain structure of the homotetrameric T4 Pnk protein and to localize essential constituents of the apparently separate active sites for the 5-kinase and 3-phosphatase activities. We characterized deletion mutants Pnk(42-301) and Pnk(1-181), which correspond to domains defined by proteolysis with chymotrypsin. Pnk(1-181) is a monomer with no 3-phosphatase and low residual 5-kinase activity. Pnk(42-301) is a dimer with no 5-kinase and low residual 3-phosphatase activity. Four classes of missense mutational effects were observed. (i) Mutations K15A, S16A, and D35A inactivated the 5-kinase but did not affect the 3-phosphatase or the tetrameric quaternary structure of T4 Pnk. 5-kinase activity was ablated by the conservative mutations K15R, K15Q, and D35N; however, kinase activity was restored by the S16T change.(ii) Mutation D167A inactivated the 3-phosphatase without affecting the 5-kinase or tetramerization. (iii) Mutation D85A caused a severe decrement in 5-kinase activity and only a modest effect on the 3-phosphatase; the nearby N87A mutation resulted in a significantly reduced 3-phosphatase activity and slightly reduced 5-kinase activity. D85A and N87A both affected the quaternary structure, resulting in a mixed population of tetramer and dimer species. (iv) Alanine mutations at 11 other conserved positions had no significant effect on either 5-kinase or 3-phosphatase activity.Polynucleotide kinase (Pnk) 1 was discovered 35 years ago in extracts of Escherichia coli infected with T-even bacteriophage (1-3). Pnk catalyzes the transfer of the ␥ phosphate from ATP or other nucleoside triphosphates to the 5Ј-OH terminus of DNA or RNA to form a 5Ј-monophosphate polynucleotide and a nucleoside diphosphate; the enzyme also phosphorylates the 5Ј-OH of nucleoside 3Ј-monophosphates (1-3). The use of T4 Pnk to label 5Ј-DNA or RNA ends with 32 P was instrumental in the subsequent identification of DNA and RNA ligases and in the development of methods for the analysis of nucleic acid structure, molecular cloning, and nucleic acid sequencing.Early biochemical studies showed that the kinase reaction is reversible (4). Although it was initially proposed that T4 Pnk acts via a ping-pong mechanism with a phosphoenzyme intermediate (4), kinetic analysis favors a sequential mechanism in which both ATP and the 5Ј-OH polynucleotide bind the enzyme prior to dissociation of either product (5). The stereochemical course of the reaction entails inversion of configuration of the transferred phosphate, indicative of an in-line mechanism in which the polynucleotide 5Ј-OH directly attacks the ␥ phosphorus of ATP (6).T4 Pnk is a bifunctional enzyme with an intrinsic 3Ј-phosphomonoesterase activity that removes 3Ј...

“…The NTP and the 5Ј-OH polynucleotide substrates enter the active site from opposite ends of the tunnel and are coordinated in their respective donor and acceptor sites to achieve phosphoryl transfer via an in-line single-displacement mechanism entailing direct attack of the 5Ј-O on the ␥-phosphorus of ATP with inversion of stereochemical configuration at the transferred phosphate (44). The T4 kinase reaction is reversible (i.e.…”

Section: Omega and Cjw1 Pnkp Have Distinct Preferences For Protrudingmentioning

confidence: 99%

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

Zhu

Yin

Journal of Biological Chemistry

2004

Coliphage T4 Pnkp is a bifunctional polynucleotide 5 -kinase/3 -phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5 -kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3 -phosphatase module with an essential acyl-phosphatase motif, DX-DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/ kinase enzymes found in mammals and fission yeast. Whereas the phage 5 -kinases differ from each other in their preferences for phosphorylation of 5 overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.Breaks in the phosphodiester backbone of genomic DNA or essential RNA molecules can lead to cell death if not repaired. Such breaks can be triggered by physical or chemical insults, enzymatic hydrolysis, exposure to cytotoxic drugs, etc. Although DNA repair pathways have been the focus of much attention in light of the connections between DNA damage, mutagenesis, and cancer, there is an emerging appreciation that distinctive pathways exist to maintain or manipulate RNA structure in response to breakage events. Viruses provided the initial sources for the identification and characterization of enzymes that rectify DNA and RNA breaks (1-12) and viral model systems continue to illuminate the mechanisms, atomic structures, and biological roles of such enzymes (13-17).When breakage of a 3Ј-5Ј phosphodiester results in the formation of 5Ј-PO 4 and 3Ј-OH termini at the break site, the ends can be rejoined to each other (or to novel partner strands) by the action of DNA-specific or RNA-specific polynucleotide ligases. However, when breakage occurs with the opposite polarity, resulting in 5Ј-OH and 3Ј-PO 4 (or 2Ј,3Ј-cyclic PO 4 ) termini, the broken ends must be "healed" before they can be sealed. Healing entails two steps: (i) hydrolysis of the 3Ј-PO 4 (or 2Ј,3Ј-cyclic phosphate) to form a 3Ј-OH and (ii) phosphorylation of the 5Ј-OH to form a 5Ј-PO 4 end.The bacteriophage T4 proteins polynucleotide kinase/phosphatase (Pnkp) and RNA ligase 1 (Rnl1) are the prototypal RNA healing and sealing enzymes (1-3, 8 -1...