Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis.
The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.
Purta
Elzbieta
E
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland.
van Vliet
Françoise
F
Tricot
Catherine
C
De Bie
Lara G
LG
Feder
Marcin
M
Skowronek
Krzysztof
K
Droogmans
Louis
L
Bujnicki
Janusz M
JM
D000595:Amino Acid Sequence; D019295:Computational Biology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C474650:YggH protein, E coli; D012359:tRNA Methyltransferases