title: Purification and comparison of native and recombinant tRNA-guanine transglycosylases from Methanosarcina acetivorans.
abstract: Many archaeal tRNAs have archaeosine (G(+)) at position 15 in the D-loop and this is thought to strengthen the tertiary interaction with C48 in the V-loop. In the first step of G(+) biosynthesis, archaeosine tRNA-guanine transglycosylase (ArcTGT)(1) catalyzes the base exchange reaction from guanine to 7-cyano-7-deazaguanine (preQ(0)). ArcTGT is classified into full-size or split types, according to databases of genomic information. Although the full-size type forms a homodimeric structure, the split type has been assumed to form a heterotetrameric structure, consisting of two kinds of peptide. However, there has been no definitive evidence for this presented to date. Here, we show that native ArcTGT could be isolated from Methanosarcina acetivorans and two peptides formed a robust complex in cells. Consequently, the two peptides function as actual subunits of ArcTGT. We also overexpressed recombinant ArcTGT in Escherichia coli cells. Product was successfully obtained by co-overexpression of the two subunits but one subunit alone was not adequately expressed in soluble fractions. This result suggests that interaction between the two subunits may contribute to the conformational stability of split ArcTGT. The values of the kinetic parameters for the recombinant and native ArcTGT were closely similar. Moreover, tRNA transcript with preQ(0) at position 15 was successfully prepared using the recombinant ArcTGT. This tRNA transcript is expected to be useful as a substrate for studies seeking the enzymes responsible for G(+) biosynthesis.
authors.affiliation: Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Nomura
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Onda
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ohno
authors.forename: Hiroki
authors.initials: H
authors.lastname: Taniguchi
authors.forename: Kaori
authors.initials: K
authors.lastname: Ando
authors.forename: Natsuhisa
authors.initials: N
authors.lastname: Oka
authors.forename: Kazuya
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokogawa
mesh_terms: D004926:Escherichia coli; D006147:Guanine / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology*; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D021122:Protein Subunits; D011994:Recombinant Proteins; D006147:Guanine; D012343:RNA, Transfer; D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase