Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues.
Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DMAPP). A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRNA transferases from various organisms revealed considerable homology, including 11 charged, 12 polar, and four aromatic amino acids that are highly conserved or conservatively substituted. Site-directed mutants were constructed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha-tubulin epitope was appended to the C-terminus of the protein to facilitate separation by immunoaffinity chromatography of overproduced mutant enzymes from coexpressed chromosomally encoded wild-type DMAPP-tRNA transferase. Steady-state kinetic constants were measured for wild-type DMAPP-tRNA transferase and the site-directed mutants using DMAPP and a 17-base RNA oligoribonucleotide corresponding to the stem-loop region of tRNA(Phe) as substrates. Substantial changes in k(cat), K(m)(DMAPP), and/or K(m)(RNA) were seen for several of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.
Soderberg
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Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
Poulter
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D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000926:Anticodon / Q000235:genetics; D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D000926:Anticodon; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012343:RNA, Transfer; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase