Structure-function analysis of the yeast NAD+-dependent tRNA 2′-phosphotransferase Tpt1 Journal Article
| Authors: | Sawaya, R.; Schwer, B.; Shuman, S. |
| Article Title: | Structure-function analysis of the yeast NAD+-dependent tRNA 2′-phosphotransferase Tpt1 |
| Abstract: | Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2′-PO4 from the splice junction to NAD+ to form ADP-ribose 1″-2″ cyclic phosphate and nicotinamide. To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo. Structure-activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the Escherichia coli ortholog KptA in complementing tpt1Δ was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30°C. These findings attest to the structural and functional conservation of Tpt1-like 2′-phosphotransferases and identify likely constituents of the active site. |
| Keywords: | unclassified drug; mutation, missense; sequence deletion; protein function; amino acid substitution; enzyme activity; structure activity relation; mutational analysis; bacteria (microorganisms); amino acid sequence; conserved sequence; molecular sequence data; sequence homology, amino acid; saccharomyces cerevisiae; escherichia coli; phosphotransferases (alcohol group acceptor); recombinant proteins; plasmids; amino acid; mutagenesis, site-directed; binding sites; yeast; saccharomyces cerevisiae proteins; alanine; transfer rna; phosphotransferase; mutagenesis; protozoa; catalyst; fungus; fungi; nad; trna splicing; archaebacterium; archaea; bacterium; protozoon; arginine; histidine; diphtheria toxin; rna splicing; amino acid motifs; escherichia coli proteins; nicotinamide adenine dinucleotide; metazoa; nicotinamide; genetic complementation test; saccharomyces; metazoon; adenosine diphosphate ribose; mutagenics; phosphoryl transfer; 2' phosphotransferase |
| Journal Title: | RNA |
| Volume: | 11 |
| Issue: | 1 |
| ISSN: | 1355-8382 |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Date Published: | 2005-01-01 |
| Start Page: | 107 |
| End Page: | 113 |
| Language: | English |
| DOI: | 10.1261/rna.7193705 |
| PUBMED: | 15611301 |
| PROVIDER: | scopus |
| PMCID: | PMC1370696 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 2" - "Export Date: 24 October 2012" - "CODEN: RNARF" - "Source: Scopus" |
