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Fold recognition, homology modeling, docking simulations, kinetics analysis and mutagenesis of ATP/CTP:tRNA nucleotidyltransferase from Methanococcus jannaschii
Author(s) -
Bujnicki Janusz M.,
Albert Megan A.,
Nelson Donald J.,
Thurlow David L.
Publication year - 2003
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10374
Subject(s) - methanococcus , docking (animal) , homology modeling , nucleotide , transfer rna , enzyme kinetics , biochemistry , stereochemistry , binding site , enzyme , active site , chemistry , site directed mutagenesis , escherichia coli , biology , rna , medicine , nursing , mutant , gene
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 μM, and the apparent K M for ATP was 21 μM. 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. Proteins 2003;51:349–359. © 2003 Wiley‐Liss, Inc.