Kinetic Mechanism of the Mg2+-dependent Nucleotidyl Transfer Catalyzed by T4 DNA and RNA Ligases

Cherepanov, Alexey V.; Vries, Simon de

doi:10.1074/jbc.m109616200

Cited by 33 publications

(56 citation statements)

References 49 publications

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“…According to the model, the catalytic metal makes direct contacts with lysine-Nζ (the nucleophile) and with the α-β bridging oxygen of ATP (the leaving group) as two of the component ligands of an octahedral metal coordination complex that also includes four waters. The model does not mandate a direct contact between the metal and nonbridging ATP α-phosphate oxygens; instead the nonbridging phosphate oxygens are depicted as accepting hydrogen bonds from two of the metalbound waters (39). The NgrRnl structures presented here militate strenuously against such a model.…”

Section: Discussionmentioning

confidence: 99%

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Unciuleac

Goldgur

Shuman

2015

Proc. Natl. Acad. Sci. U.S.A.

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ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO 4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway-covalent ligase-(lysyl- 2+ complexhighlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound "catalytic" metal coordination complex lowers the pK a of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β-and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes.NζRNA repair | covalent nucleotidyltransferase | lysyl-AMP B iochemically diverse RNA repair systems rely on RNA ligases to maintain or manipulate RNA structure in response to purposeful RNA breakage events (1-5). RNA breaks destined for repair are inflicted by sequence-specific or structure-specific endoribonucleases during physiological RNA processing (e.g., tRNA splicing; kinetoplast mRNA editing) and under conditions of cellular stress (e.g., virus infection, starvation, unfolded protein response). RNA cleavage can occur either: (i) by a transesterification mechanism (generally metal-independent) that yields 2′,3′-cyclic-PO 4 and 5′-OH ends; or (ii) via a hydrolytic mechanism (typically metaldependent) that leaves 3′-OH and 5′-PO 4 ends. RNA repair enzymes capable of sealing 2′,3′-cyclic-PO 4 /5′-OH breaks or 3′-OH/5′-PO 4 breaks are distributed widely in all phylogenetic domains of life.ATP-dependent RNA ligases join 3′-OH and 5′-PO 4 RNA termini via a series of three nucleotidyl transfer steps similar to those of DNA ligases (6). In step 1, RNA ligase reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate plus pyrophosphate. In step 2, AMP is transferred from ligase-adenylate to the 5′-PO 4 RNA end to form an RNA-adenylate intermediate, AppRNA. In step 3, ligase catalyzes attack by an RNA 3′-OH on the RNA-adenylate to seal the two ends via a phosphodiester bond and release AMP.The autoadenylylation reaction of RNA ligases is performed by a nucleotidyltransferase (NTase) domain that is conserved in ATP-dependent and NAD + -dependent DNA ligases and GTPdependent mRNA capping enzymes (6, 7). The NTase domain includes six peptide motifs (I, Ia, III, IIIa, IV, and V) that form the nucleotide-bin...

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Section: Discussionmentioning

confidence: 99%

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Unciuleac

Goldgur

Shuman

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Studies on human RtcB and Archease suggest that Archease facilitates the guanylylation of RtcBq after a single turnover, enabling another round of catalysis . RtcB uses a two-manganese mechanism during catalysis (Desai et al 2013), which is analogous to the two-magnesium mechanism used by classical ligases (Cherepanov and de Vries 2002). The presence of an essential metal-binding site on the exterior of Archease at a tip, has led us to suggest that it might function to reach into and position Mn(II) ions within the RtcB active site.…”

Section: Discussionmentioning

confidence: 99%

Coevolution of RtcB and Archease created a multiple-turnover RNA ligase

Desai

Beltrame

Raines

2015

RNA

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RtcB is a noncanonical RNA ligase that joins either 2 ′ ,3 ′ -cyclic phosphate or 3 ′ -phosphate termini to 5 ′ -hydroxyl termini. The genes encoding RtcB and Archease constitute a tRNA splicing operon in many organisms. Archease is a cofactor of RtcB that accelerates RNA ligation and alters the NTP specificity of the ligase from Pyrococcus horikoshii. Yet, not all organisms that encode RtcB also encode Archease. Here we sought to understand the differences between Archease-dependent and Archease-independent RtcBs so as to illuminate the evolution of Archease and its function. We report on the Archeasedependent RtcB from Thermus thermophilus and the Archease-independent RtcB from Thermobifida fusca. We find that RtcB from T. thermophilus can catalyze multiple turnovers only in the presence of Archease. Remarkably, Archease from P. horikoshii can activate T. thermophilus RtcB, despite low sequence identity between the Archeases from these two organisms. In contrast, RtcB from T. fusca is a single-turnover enzyme that is unable to be converted into a multiple-turnover ligase by Archease from either P. horikoshii or T. thermophilus. Thus, our data indicate that Archease likely evolved to support multiple-turnover activity of RtcB and that coevolution of the two proteins is necessary for a functional interaction.

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“…Nucleophilic attack on the ␣-phosphorus of ATP results in cleavage of the triphosphate moiety, formation of the enzyme-AMP -amino lysyl phosphoramidate, and release of the pyrophosphate (10). This reaction is reversible; we have shown that the dimagnesium ATP⅐Mg 2 form is the true substrate for the transfer of adenylyl moiety to the ligase (11). In the reverse reaction, DNA ligase uses the mono-magnesium form of pyrophosphate to synthesize ATP.…”

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confidence: 95%

“…In the stopped-flow instrument, this reaction is observed as a monophasic process with an activation energy of 16.2 kcal/mol (12). For T4 DNA ligase, ATP and pyrophosphate dissociation constants are Ͻ2 and 30 M, respectively (11). Thus, at the millimolar enzyme concentration used in this work, chemical equilibrium is shifted toward the protein-ligand complexes.…”

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confidence: 96%

“…In the reverse reaction, DNA ligase uses the mono-magnesium form of pyrophosphate to synthesize ATP. In the absence of Mg 2ϩ , the enzyme binds ATP noncovalently; when Mg 2ϩ is added, nucleotidyl transfer starts under pseudo-first-order conditions (11). In the stopped-flow instrument, this reaction is observed as a monophasic process with an activation energy of 16.2 kcal/mol (12).…”

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confidence: 99%

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The associative nature of adenylyl transfer catalyzed by T4 DNA ligase

Cherepanov

Doroshenko²,

Matysik³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD ؉ and assistance of a divalent metal cofactor Mg 2؉ . Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover 31 P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg 2؉ , and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4 -5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.chemical movie ͉ nucleotidyl transfer ͉ structural reaction kinetics ͉ time-resolved cryo-magic-angle-spinning NMR ͉ transition state U nderstanding chemical mechanics of biocatalysis is a fundamental goal of life sciences. With the development of high-resolution x-ray diffraction analysis and solution NMR a large number of protein structures in the resting state have been solved, giving knowledge on how proteins look, e.g., the detailed view of protein architecture on the primary, secondary, tertiary, and quaternary structure levels. Further insight is coming with studies on how proteins work, e.g., by observing changes of the protein structure in the course of a chemical reaction-recording a molecular movie. Femtosecond laser pulses, molecular beams and ultrafast electron diffraction are used in (in)organic chemistry for monitoring breaking and forming of chemical bonds in real time (1). In biochemistry, kinetic crystallography is used to record molecular movies, an approach that combines starting and stopping the reaction in a protein crystal with x-ray data acquisition at low temperatures (2-8). The successful outcome of a time-resolved x-ray experiment depends as much on a prompt triggering of the reaction as on preparing well diffracting protein crystals. Here we introduce time-resolved lowtemperature magic-angle-spinning (cryo-MAS) NMR spectroscopy as a complementary ''noninvasive'' technique to study catalytic dynamics of biochemical reactions. It combines phototriggering and freeze-trapping with real-time monitoring of chemical transformations and requires neither protein crystallization nor high intensity penetrating radiation beams. We have assayed the nucleotidyl transfer reaction catalyzed by DNA ligase from bacteriophage T4, a Mg 2ϩ -and AT...

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Kinetic Mechanism of the Mg2+-dependent Nucleotidyl Transfer Catalyzed by T4 DNA and RNA Ligases

Cited by 33 publications

References 49 publications

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

Coevolution of RtcB and Archease created a multiple-turnover RNA ligase

The associative nature of adenylyl transfer catalyzed by T4 DNA ligase

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