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

Biochemistry
T Soderberg, C D Poulter

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.

Citations

Jun 27, 2002·Trends in Pharmacological Sciences·Niki Zacharias, Dennis A Dougherty
Sep 3, 2013·Journal of the American Chemical Society·Bradley J LandgrafSquire J Booker
Mar 28, 2009·The Journal of Organic Chemistry·C Dale Poulter
Oct 15, 2008·Proceedings of the National Academy of Sciences of the United States of America·Chun Zhou, Raven H Huang
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Nov 1, 2005·EcoSal Plus·Glenn R Björk, Tord G Hagervall
Jul 3, 2021·Microorganisms·Jitka Frébortová, Ivo Frébort

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