tRNA-guanine transglycosylase from Escherichia coli is a zinc metalloprotein. Site-directed mutagenesis studies to identify the zinc ligands.
tRNA-guanine transglycosylase (TGT) from Escherichia coli catalyzes the exchange of the queuine precursor, preQ1, into tRNA as part of the biosynthetic pathway for the posttranscriptionally modified base, queuine. No significant sequence homologies exist between TGT and any of the proteins in the GenBank database. However, an unusual arrangement of cysteine residues was observed upon manual examination of the TGT sequence. Comparison of this sequence (residues 302-321) revealed similarities to structural zinc-binding motifs in proteins of known structure [Jaffe (1993) Comments Inorg. Chem. 15, 67-93]. Within this region of the TGT sequence, there are six residues (four cysteines and two histidines), any four of which could serve as the ligands to the zinc. We report here that wild-type TGT contains ca. 0.8 mol of zinc/mol of subunit, determined by atomic emission spectrometry. In order to determine which enzyme residues are serving as the ligands to the zinc, site-directed mutagenesis studies have been performed. Gross structural probes (native PAGE and CD spectra), enzyme activity assays, and tRNA-binding assays indicate that cysteines 302, 304, and 307 and histidine 317 are the ligands to the zinc. These results also suggest that the zinc site is necessary for TGT homotrimer formation and for tRNA binding.
Chong
S
S
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Curnow
A W
AW
Huston
T J
TJ
Garcia
G A
GA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010618:Phenanthrolines; D017433:Protein Structure, Secondary; D015032:Zinc / Q000378:metabolism*
D017931:DNA Primers; D008024:Ligands; D010618:Phenanthrolines; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase; D015032:Zinc; C025205:1,10-phenanthroline