Fold recognition, homology modeling, docking simulations, kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii.
ATP/CTP:tRNA nucleotidyltransferases (NTases) and poly(A) polymerases (PAPs) belong to the same superfamily and their catalytic domains are remotely related. Based on the results of fold-recognition analysis and comparison of secondary structure patterns, we predicted that these two NTase families share three domains, corresponding to "palm," "fingers," and "fingernails" in the PAP crystal structure. A homology model of tRNA NTase from Methanococcus jannaschii was constructed. Energy minimization calculations of enzyme-nucleotide complexes and computer-aided docking of nucleotides onto the enzyme's surface were carried out to explore possible ATP and CTP binding sites. Theoretical models were used to guide experimental analysis. Recombinant His-tagged enzyme was expressed in Escherichia coli, and kinetic properties were characterized. The apparent K(M) for CTP was determined to be 38 microM, and the apparent K(M) for ATP was 21 microM. Three mutations of basic amino acids to alanine were created in a highly conserved region predicted to be in the vicinity of the nucleotide binding site. A deletion was also constructed to remove the C-terminal structural domain defined by the model; it retained about 1% of wild type enzymatic activity using CTP as co-substrate, confirming that detectable catalytic activity is exhibited by the N-terminal domain, as defined by the model. Our results suggest a mechanism of differential ATP and CTP binding, which explains how the tRNA NTase, having only one catalytic site, utilizes different nucleotide triphosphates depending on the nature of the tRNA substrate.
Bujnicki
Janusz M
JM
Bioinformatics Laboratory, International Institute of Molecular and Cell Biology, Warsaw, Poland. iamb@genesilico.pl
Albert
Megan A
MA
Nelson
Donald J
DJ
Thurlow
David L
DL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003198:Computer Simulation; D003570:Cytidine Triphosphate / Q000378:metabolism; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase