Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli.
An Escherichia coli mutant described by Noguchi et al. [Noguchi, S., et al. (1982) J. Biol. Chem. 275, 6544-6550] contains tRNA lacking the hypermodified wobble nucleoside queuosine (Q) due to an inactive tRNA-guanine transglycosylase (TGT). TGT catalyzes the posttranscriptional base exchange of the Q precursor preQ1 with the genetically encoded guanine in tRNA(Asp,Asn,His,Tyr). The mutant tgt gene was cloned and sequenced; it contained a single point mutation resulting in the change of serine 90 to phenylalanine. Overexpression of the mutant gene yielded TGT(S90F) that showed a reduced solubility and did not purify in the same fashion as the wild-type enzyme. TGT(S90F) has no detectable enzymic activity. To determine whether serine 90 performs a catalytic role in the TGT reaction or whether the loss of activity was caused solely by a conformational change of the enzyme, we used site-specific mutagenesis to construct serine-to-alanine (S90A) and serine-to-cysteine (S90C) mutants. Both S90A and S90C mutants were purified in a manner identical to that used for the wild-type enzyme. SDS-PAGE of dimethyl suberimidate-cross-linked mutants showed a pattern identical to that of the wild-type TGT, indicative of a trimeric quaternary structure. Native PAGE of wild-type and mutant TGTs in the absence and presence of substrate tRNA exhibited band shifts indicating that both mutants retain the ability to bind tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Reuter
K
K
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Chong
S
S
Ullrich
F
F
Kersten
H
H
Garcia
G A
GA
D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010430:Pentosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D012694:Serine; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase