Covalent catalysis in nucleotidyl transfer reactions: Essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases Journal Article
| Authors: | Shuman, S.; Liu, Y.; Schwer, B. |
| Article Title: | Covalent catalysis in nucleotidyl transfer reactions: Essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases |
| Abstract: | Formation of the 5′ cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5′ terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltranferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases. |
| Keywords: | nonhuman; comparative study; gene expression; gene library; genetic vectors; cloning, molecular; amino acid sequence; conserved sequence; molecular sequence data; sequence homology, amino acid; saccharomyces cerevisiae; dna viruses; eukaryota; recombinant proteins; base sequence; mutagenesis, site-directed; catalysis; sequence homology; point mutation; protein family; polydeoxyribonucleotide synthase; site directed mutagenesis; guanosine triphosphate; genetic conservation; enzyme mechanism; schizosaccharomyces; schizosaccharomyces pombe; rna polymerase; capped rna; rna capping; genetic complementation test; nucleotidyltransferases; viruses; genes, fungal; gene isolation; guanosine phosphate; human; priority journal; article |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 91 |
| Issue: | 25 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1994-12-06 |
| Start Page: | 12046 |
| End Page: | 12050 |
| Language: | English |
| PROVIDER: | scopus |
| PMCID: | PMC45373 |
| PUBMED: | 7991582 |
| DOI: | 10.1073/pnas.91.25.12046 |
| DOI/URL: | |
| Notes: | Export Date: 14 January 2019 -- Article -- CODEN: PNASA C2 -- Source: Scopus |
