SEQUENCE-ANALYSIS AND OVEREXPRESSION OF THE ZYMOMONAS-MOBILIS TGT GENE ENCODING TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE - PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME

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 .