SEQUENCE-ANALYSIS AND OVEREXPRESSION OF THE ZYMOMONAS-MOBILIS TGT GENE ENCODING TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE - PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME
K. Reuter et R. Ficner, SEQUENCE-ANALYSIS AND OVEREXPRESSION OF THE ZYMOMONAS-MOBILIS TGT GENE ENCODING TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE - PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME, Journal of bacteriology, 177(18), 1995, pp. 5284-5288
tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of
the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the pos
ttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deaz
aguanine (preQ(1)) with the genetically encoded guanine in the anticod
on of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr). A partially seque
nced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobil
is chromosome shows strong homology to the tgt gene of Escherichia col
i (K, B, Shark and T, Conway, FEMS Microbiol, Lett, 96:19-26, 1992). W
e showed that this gene is able to complement the tgt mutation in E. c
oli SJ1505, and we determined its complete sequence. Four start codons
were possible for this gene, resulting in proteins of 386 to 399 amin
o acids (M(r) 42,800 to 44,300) showing 60.4% sequence identity with T
gt from E. coli, The smallest of the four possible reading frames, whi
ch was still extended at its 5' end compared with the E. coli tgt gene
, was overexpressed in E. coli. The gene product was purified to homog
eneity and was biochemically characterized. The kinetical parameters w
ere virtually identical to those published for the E. coli enzyme. In
contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobi
lis Tgt was found to be a monomer according to gel filtration, In this
study, it was shown that the formation of homotrimers by the E. coli
enzyme is readily reversible and is dependent on protein concentration
.