title: The Candida albicans gene for mRNA 5-cap methyltransferase: identification of additional residues essential for catalysis.
abstract: The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N7-)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C. albicans ABD1 (CaABD1). When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase activity in vitro and rescued S. cerevisiae abd1delta null mutants, indicating that CaABD1 specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5'-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality in vivo, whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif.
authors.affiliation: Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan1.
authors.forename: Toshiko
authors.initials: T
authors.lastname: Yamada-Okabe
authors.affiliation: Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan2.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Mio
authors.affiliation: Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan1.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kashima
authors.affiliation: Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan1.
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Matsui
authors.affiliation: Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan2.
authors.forename: Mikio
authors.initials: M
authors.lastname: Arisawa
authors.affiliation: Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan2.
authors.forename: Hisafumi
authors.initials: H
authors.lastname: Yamada-Okabe
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009713:Nucleotidyltransferases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D000098556:mRNA Guanylyltransferases
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases