Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12.
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 tRNAPhe, the anticodon stem-loop (ACSLPhe) of tRNAPhe, 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 tRNAPhe binding. MiaA had a Kmapp (tRNA substrates) approximately 3 nM, which is orders of magnitude lower than that of other purified tRNA modification enzymes, a Kmapp (DMAPP) = 632 nM, and a kcatapp = 0.44 s-1. MiaA activity was minimally affected by other modifications or nonsubstrate tRNA species present in bulk tRNA isolated from a miaA mutant. MiaA modified ACSLPhe with a kcatapp/Kmapp substrate specificity about 17-fold lower than that for intact tRNAPhe, 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 microM concentration) and is present in a catalytic excess. However, MiaA activity was strongly competitively inhibited for DMAPP by ATP and ADP (Kiapp = 0.06 microM), 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 (Kdapp approximately 0.07 microM).
Leung
H C
HC
Department of Microbiology and Molecular Genetics, University of Texas Houston Medical School, Houston, Texas 77030-1501, USA.
Chen
Y
Y
Winkler
M E
ME
D019883:Alkyl and Aryl Transferases; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D045782:Hemiterpenes; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C043060:3,3-dimethylallyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C023197:tRNA isopentenyltransferase