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Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate

Nature Structural & Molecular Biology volume 10, pages 781–788 (2003)Cite this article

A Corrigendum to this article was published on 01 July 2004

Abstract

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 Å 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.

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Figure 1: Trapping of a TGT–RNA covalent intermediate.
Figure 2: Alignment of TGT sequences from different organisms.
Figure 3: Overall structure of the trapped covalent intermediate.
Figure 4: Conformational changes in RNA and TGT.
Figure 5: The active site and molecular basis of substrate specificity.
Figure 6: Enzymatic activities of TGT mutants.
Figure 7: The chemical reaction carried out in crystal and the resulting structural changes.

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Acknowledgements

We are grateful to T. Conway (University of Oklahoma) for a plasmid encoding the Z. mobilis TGT and to P. Tyler (Industrial Research Ltd., New Zealand) for providing the initial 9dzG. We thank the staffs of beamlines 14-BMC (K. Brister, T. Teng and R. Pahl) and 19-ID (S. Ginell, A. Joachimiak and Y. Kim) at APS for their support during data collections; Y. Elias, K. Phannach and other members of the Huang research group; and D. Kranz, R. Switzer and J. Gerlt for many helpful discussions and critical reading of the manuscript. The work was supported by start-up funds from the University of Illinois and a grant from the National Institutes of Health.

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  1. Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, 61801, Illinois, USA

    Wei Xie, Xianjun Liu & Raven H Huang

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Correspondence to Raven H Huang.

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Xie, W., Liu, X. & Huang, R. Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat Struct Mol Biol 10, 781–788 (2003). https://doi.org/10.1038/nsb976

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