Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: a binding mechanism for recombinant enzyme.
Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine. The enzyme, encoded by the miaA gene, was overproduced and purified to apparent homogeneity in three steps by ion-exchange (DE52 and Mono-Q) and size exclusion chromatography. Affinity-tagged DMAPP-tRNA transferase containing a C-terminal tripeptide alpha-tubulin epitope also was overproduced and purified to apparent homogeneity in two steps by ion-exchange and immunoaffinity chromatography. Addition of the C-terminal tripeptide alpha-tubulin epitope to DMAPP-tRNA transferase did not affect the activity of the enzyme. Undermodified tRNA(Phe) used as substrate in the DMAPP-tRNA transferase-catalyzed reaction was isolated and purified from an overexpressing clone in a miaA deficient strain of E. coli. Active recombinant E. coli DMAPP-tRNA transferase is monomeric. The enzyme transferred the dimethylallyl moiety of DMAPP to A37, located adjacent to the anticodon in undermodified tRNA(Phe). The enzyme required Mg2+ for activity and exhibited a broad pH optimum. Michaelis constants for tRNA(Phe) and DMAPP are 96 +/- 11 nM and 3.2 +/- 0.5 microM, respectively, and Vmax = 0.83 +/- 0.02 micromol min-1 mg-1. DMAPP-tRNA transferase bound tRNA(Phe) with a dissociation constant of 5.2 +/- 1.2 nM. In contrast, DMAPP did not bind to the enzyme in the absence of tRNA. However, DMAPP was bound with a dissociation constant of 3.4 +/- 0.6 microM in the presence of a minihelix analogue of the anticodon stem-loop of tRNA(Phe) where the base corresponding to A37 was replaced by inosine. These results suggest an ordered sequential mechanism for substrate binding.
Moore
J A
JA
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Poulter
C D
CD
D019883:Alkyl and Aryl Transferases; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology*; D045782:Hemiterpenes; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009943:Organophosphorus Compounds / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; C043060:3,3-dimethylallyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase