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

2015 ◽  
Vol 112 (45) ◽  
pp. 13868-13873 ◽  
Author(s):  
Mihaela-Carmen Unciuleac ◽  
Yehuda Goldgur ◽  
Stewart Shuman
Keyword(s):  
Reaction Pathway ◽  
Domain Architecture ◽  
Coordination Complex ◽  
Metal Coordination ◽  
Rna Repair ◽  
Rna Ligase ◽  
Dna Ligases ◽  
Catalytic Metal ◽  
Rna Capping ◽  
Unique Domain

ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO4 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-Nζ)–AMP•Mn2+ intermediate; ligase•ATP•(Mn2+)2 Michaelis complex; and ligase•Mn2+ complex—highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound “catalytic” metal coordination complex lowers the pKa 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.

scholarly journals Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

2019 ◽  
Vol 47 (14) ◽  
pp. 7147-7162 ◽  
Author(s):  
Adele Williamson ◽  
Hanna-Kirsti S Leiros
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Bond Formation ◽  
Strand Break ◽  
Nucleophilic Attack ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Dna Ligases ◽  
Catalytic Metal ◽  
Ternary Structure

Abstract DNA ligases join adjacent 5′ phosphate (5′P) and 3′ hydroxyl (3′OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase–DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3′OH terminus for nucleophilic attack on the 5′P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

scholarly journals Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism

2018 ◽  
Vol 19 (12) ◽  
pp. 3839 ◽  
Author(s):  
Daniel Peters ◽  
Laura Kay ◽  
Jeyanthy Eswaran ◽  
Jeremy Lakey ◽  
Meera Soundararajan
Keyword(s):  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Domain Architecture ◽  
Subcellular Localisation ◽  
Cellular Processes ◽  
Calcium Sensing ◽  
And Function ◽  
Arginine Finger ◽  
Catalytic Functions ◽  
Unique Domain

Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are “atypical” Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues (equivalent to Ras Y32, T35, G60 and Q61), the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be “relic” domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an “internal arginine finger”, allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms.

scholarly journals Evolution of Parallel Spindles Like genes in plants and highlight of unique domain architecture#

2011 ◽  
Vol 11 (1) ◽  
Author(s):  
Riccardo Aiese Cigliano ◽  
Walter Sanseverino ◽  
Gaetana Cremona ◽  
Federica M Consiglio ◽  
Clara Conicella
Keyword(s):  
Domain Architecture ◽  
Parallel Spindles ◽  
Unique Domain

scholarly journals A Novel Peroxiredoxin Activity Is Located within the C-Terminal End of Rhodospirillum rubrum Adenylyltransferase

2007 ◽  
Vol 190 (1) ◽  
pp. 434-437 ◽  
Author(s):  
Anders Jonsson ◽  
Pedro Filipe Teixeira ◽  
Stefan Nordlund
Keyword(s):  
Glutamine Synthetase ◽  
Rhodospirillum Rubrum ◽  
Domain Architecture ◽  
Carboxyl Terminal ◽  
Unique Domain ◽  
First Time

ABSTRACT Adenylyltransferase (GlnE) catalyzes the reversible adenylylation of glutamine synthetase. In this report we present, for the first time, evidence for a peroxiredoxin activity of Rhodospirillum rubrum GlnE, through the carboxyl-terminal AhpC/thiol-specific antioxidant (TSA) domain. The combination of GlnE and AhpC/TSA domains within the same polypeptide constitutes a unique domain architecture that has not previously been identified among proteobacteria.

scholarly journals Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

2015 ◽  
Vol 197 (22) ◽  
pp. 3616-3624 ◽  
Author(s):  
William P. Maughan ◽  
Stewart Shuman
Keyword(s):  
Specific Activity ◽  
Myxococcus Xanthus ◽  
Rna Repair ◽  
Content Type ◽  
Rna Ligase ◽  
E Coli ◽  
Dna And Rna ◽  
Genes Encoding ◽  
The Many ◽  
Bacterial Proteomes

ABSTRACTEscherichia coliRtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3′-PO4ends to 5′-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA3′pp5′G intermediates.E. coliRtcB also transfers GMP to a DNA 3′-PO4end to form a stable “capped” product, DNA3′pp5′G. RtcB homologs are found in a multitude of bacterial proteomes, and many bacteria have genes encoding two or more RtcB paralogs; an extreme example isMyxococcus xanthus, which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of threeM. xanthusRtcB paralogs. We found thatM. xanthusRtcB1 resemblesE. coliRtcB in its ability to perform intra- and intermolecular sealing of aHORNAp substrate and capping of a DNA 3′-PO4end.M. xanthusRtcB2 can spliceHORNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast,M. xanthusRtcB3 is distinctively feeble at ligating theHORNAp substrate, although it readily caps a DNA 3′-PO4end. The novelty ofM. xanthusRtcB3 is its capacity to cap DNA and RNA 5′-PO4ends to form GppDNA and GppRNA products, respectively. As such, RtcB3 joins a growing list of enzymes (including RNA 3′-phosphate cyclase RtcA and thermophilic ATP-dependent RNA ligases) that can cap either end of a polynucleotide substrate. GppDNA formed by RtcB3 can be decapped to pDNA by the DNA repair enzyme aprataxin.IMPORTANCERtcB enzymes comprise a widely distributed family of RNA 3′-PO4ligases distinguished by their formation of 3′-GMP-capped RNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire ofE. coliRtcB are well studied, but it is unclear whether its properties apply to the many bacteria that have genes encoding multiple RtcB paralogs. A comparison of the biochemical activities of threeM. xanthusparalogs, RtcB1, RtcB2, and RtcB3, shows that not all RtcBs are created equal. The standout findings concern RtcB3, which is (i) inactive as an RNA 3′-PO4ligase but adept at capping a DNA 3′-PO4end and (ii) able to cap DNA and RNA 5′-PO4ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications.

A Photoluminescent Metal Coordination Complex Constructed from Hydrothermal in situ Generated Quinoline-monoacyl­hydrazidate Ligand

2015 ◽  
Vol 642 (1) ◽  
pp. 20-24 ◽  
Author(s):  
Qing-Feng Yang ◽  
Hong-Cun Bai ◽  
Bing Li ◽  
Min Luo ◽  
Juan Jin ◽  
...  
Keyword(s):  
Coordination Complex ◽  
Metal Coordination

Chromogenic detection of Sarin by discolouring decomplexation of a metal coordination complex

2013 ◽  
Vol 49 (79) ◽  
pp. 8946 ◽  
Author(s):  
Lucie Ordronneau ◽  
Alexandre Carella ◽  
Miroslav Pohanka ◽  
Jean-Pierre Simonato
Keyword(s):  
Coordination Complex ◽  
Metal Coordination

scholarly journals Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases

2017 ◽  
Vol 114 (10) ◽  
pp. 2592-2597 ◽  
Author(s):  
Mihaela-Carmen Unciuleac ◽  
Yehuda Goldgur ◽  
Stewart Shuman
Keyword(s):  
Coordination Complex ◽  
Antibacterial Drug ◽  
Rna Ligase ◽  
Repair Enzymes ◽  
Nicotinamide Mononucleotide ◽  
T4 Rna Ligase ◽  
Atomic Interactions ◽  
Leaving Group ◽  
Catalytic Mechanisms ◽  
Antibacterial Drug Target

Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3′-OH and 5′-PO4DNA or RNA ends. Ligases react with ATP or NAD+and a divalent cation cofactor to form a covalent enzyme-(lysine-Nζ)–adenylate intermediate. Here, we report crystal structures of the founding members of the ATP-dependent RNA ligase family (T4 RNA ligase 1; Rnl1) and the NAD+-dependent DNA ligase family (Escherichia coliLigA), captured as their respective Michaelis complexes, which illuminate distinctive catalytic mechanisms of the lysine adenylylation reaction. The 2.2-Å Rnl1•ATP•(Mg2+)2structure highlights a two-metal mechanism, whereby: a ligase-bound “catalytic” Mg2+(H2O)5coordination complex lowers the pKaof the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; a second octahedral Mg2+coordination complex bridges the β and γ phosphates; and protein elements unique to Rnl1 engage the γ phosphate and associated metal complex and orient the pyrophosphate leaving group for in-line catalysis. By contrast, the 1.55-Å LigA•NAD+•Mg2+structure reveals a one-metal mechanism in which a ligase-bound Mg2+(H2O)5complex lowers the lysine pKaand engages the NAD+α phosphate, but the β phosphate and the nicotinamide nucleoside of the nicotinamide mononucleotide (NMN) leaving group are oriented solely via atomic interactions with protein elements that are unique to the LigA clade. The two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.

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