Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate.
Prokaryotic tRNA guanine transglycosylase (TGT) catalyzes replacement of guanine (G) by 7-aminomethyl-7-deazaguanine (PreQ1) at the wobble position of four specific tRNAs. Addition of 9-deazaguanine (9dzG) to a reaction mixture of Zymomonas mobilis TGT and an RNA substrate allowed us to trap, purify and crystallize a chemically competent covalent intermediate of the TGT-catalyzed reaction. The crystal structure of the TGT-RNA-9dzG ternary complex at a resolution of 2.9 A reveals, unexpectedly, that RNA is tethered to TGT through the side chain of Asp280. Thus, Asp280, instead of the previously proposed Asp102, acts as the nucleophile for the reaction. The RNA substrate adopts an unusual conformation, with four out of seven nucleotides in the loop region flipped out. Interactions between TGT and RNA revealed by the structure provide the molecular basis of the RNA substrate requirements by TGT. Furthermore, reaction of PreQ1 with the crystallized covalent intermediate provides insight into the necessary structural changes required for the TGT-catalyzed reaction to occur.
Xie
Wei
W
Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Liu
Xianjun
X
Huang
Raven H
RH
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000737:chemistry*; D011487:Protein Conformation; D013379:Substrate Specificity; D017355:Zymomonas / Q000737:chemistry / Q000201:enzymology / Q000235:genetics
D010430:Pentosyltransferases; C020837:queuine tRNA-ribosyltransferase