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Volume 272, Number 20, Issue of May 16, 1997 pp. 13073-13083
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Substrate Recognition by the MiaA tRNA Prenyltransferase Modification Enzyme of Escherichia coli K-12

(Received for publication, January 6, 1997, and in revised form, February 18, 1997)

From the Department of Microbiology and Molecular Genetics, University of Texas Houston Medical School, Houston, Texas 77030-1501

We purified polyhistidine (His₆)-tagged and native Escherichia coli MiaA tRNA prenyltransferase, which uses dimethylallyl diphosphate (DMAPP) to isopentenylate A residues adjacent to the anticodons of most tRNA species that read codons starting with U residues. Kinetic and binding studies of purified MiaA were performed with several substrates, including synthetic wild-type tRNA^Phe, the anticodon stem-loop (ACSL^Phe) of tRNA^Phe, and bulk tRNA isolated from a miaA mutant. Gel filtration shift and steady-state kinetic determinations showed that affinity-purified MiaA had the same properties as native MiaA and was completely active for tRNA^Phe binding. MiaA had a K_m^app (tRNA substrates) 3 nM, which is orders of magnitude lower than that of other purified tRNA modification enzymes, a K_m^app (DMAPP) = 632 nM, and a k_cat^app = 0.44 s¹. MiaA activity was minimally affected by other modifications or nonsubstrate tRNA species present in bulk tRNA isolated from a miaA mutant. MiaA modified ACSL^Phe with a k_cat^app/K_m^app substrate specificity about 17-fold lower than that for intact tRNA^Phe, mostly due to a decrease in apparent substrate binding affinity. Quantitative Western immunoblotting showed that MiaA is an abundant protein in exponentially growing bacteria (660 monomers per cell; 1.0 µM concentration) and is present in a catalytic excess. However, MiaA activity was strongly competitively inhibited for DMAPP by ATP and ADP (K_i^app = 0.06 µM), suggesting that MiaA activity is inhibited substantially in vivo and that DMAPP may bind to a conserved P-loop motif in this class of prenyltransferases. Band shift, filter binding, and gel filtration shift experiments support a model in which MiaA tRNA substrates are recognized by binding tightly to MiaA multimers possibly in a positively cooperative way (K_d^app 0.07 µM).

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