Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5′-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick Journal Article
| Authors: | Raymond, A.; Shuman, S. |
| Article Title: | Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5′-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick |
| Abstract: | Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3′-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5′-PO4. Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure - activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes. © 2007 Oxford University Press. |
| Keywords: | controlled study; unclassified drug; nonhuman; animal cell; amino acid substitution; serine; carboxy terminal sequence; enzyme activity; structure activity relation; structure-activity relationship; mutational analysis; evolution, molecular; rna; bacterial proteins; amino acid sequence; molecular sequence data; amino terminal sequence; eukaryota; nucleotide sequence; protein structure, tertiary; threonine; alanine; sequence homology; glutamine; lysine; rna ligase (atp); glycine; arginine; histidine; phenylalanine; amino acid motifs; mutant; adenylyltransferase; adenosine phosphate; adenylation; phosphodiester; nucleotidyltransferase; ester; deinococcus radiodurans; deinococcus; rna ligase; transferase; eukaryotic cells |
| Journal Title: | Nucleic Acids Research |
| Volume: | 35 |
| Issue: | 3 |
| ISSN: | 0305-1048 |
| Publisher: | Oxford University Press |
| Date Published: | 2007-02-01 |
| Start Page: | 839 |
| End Page: | 849 |
| Language: | English |
| DOI: | 10.1093/nar/gkl1090 |
| PUBMED: | 17204483 |
| PROVIDER: | scopus |
| PMCID: | PMC1807946 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 17 November 2011" - "CODEN: NARHA" - "Molecular Sequence Numbers: GENBANK: CAE76396, EAL61744, XP_367846, XP_380758;" - "Source: Scopus" |
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