Escherichia coli dimethylallyl diphosphate : tRNA dimethylallyltransferase: Site-directed mutagenesis of highly conserved residues

Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transfe rase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DM APP). A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRN A transferases from various organisms revealed considerable homology, inclu ding II charged, 12 polar, and four aromatic amino acids that are highly co nserved or conservatively substituted. Site-directed mutants were construct ed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha -tubuli n epitope was appended to the C-terminus of the protein to facilitate separ ation 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 oligoribonucleo tide corresponding to the stem-loop region of tRNA(Phe) as substrates. Subs tantial changes in k(cat), K-m(DMAPP), and/or K-m(RNA) were seen for severa l of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.