PMID:11327834
| Citation |
Soderberg, T and Poulter, CD (2001) Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues. Biochemistry 40:1734-40 |
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| Abstract |
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. |
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| Keywords |
Alkyl and Aryl Transferases/chemistry; Alkyl and Aryl Transferases/genetics; Amino Acid Sequence; Amino Acid Substitution/genetics; Anticodon/genetics; Catalysis; Circular Dichroism; Conserved Sequence/genetics; Enzyme Activation/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Transfer/chemistry; RNA, Transfer/genetics; Recombinant Proteins/chemical synthesis; Recombinant Proteins/genetics; Substrate Specificity/genetics |
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