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Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

Abstract

A previously determined crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state–like arrangement based on validoxylamine A 6′-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion–like transition state. Leaving group 18O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Brønsted analysis of nucleophiles and Taft analysis highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quantitative data substantiate this unusual enzymatic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.

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Figure 1: The reaction catalyzed by trehalose-6-phosphate synthase (OtsA).
Figure 2: Linear free energy relationships of OtsA variants.
Figure 3: A schematic diagram depicting the profile (energy versus coordinate) of OtsA-catalyzed reaction.
Figure 4: LFER of acceptor kinetics determined using the varied modified acceptors.

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Acknowledgements

We thank the Bill and Melinda Gates Foundation, the Samsung Fellowship and the Biotechnology and Biological Sciences Research Council for financial support. R. Zhang and S.G. Withers (University of British Columbia) are acknowledged for providing 2-deoxy-2,2-difluoroglucose; R. Gibson (currently at the University of Liverpool) is thanked for initial construction of some OtsA mutants. B.G.D. and G.J.D. are both Royal Society Wolfson Research Merit Award recipients, and B.G.D. is supported by an Engineering and Physical Sciences Research Council Life Sciences Interface Platform grant.

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S.S.L. and B.G.D. designed the experiments. A.I. provided mutant otsA plasmids. S.S.L., S.Y.H. and J.C.E. expressed the mutants. S.S.L. and J.C.E. performed the kinetic measurements. S.S.L. performed all other experiments. S.S.L., B.G.D. and G.J.D. analyzed the experiments. B.G.D., S.S.L. and G.J.D. wrote the manuscript.

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Correspondence to Benjamin G Davis.

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The authors declare no competing financial interests.

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Supplementary Figs. 1–3, Tables 1 and 3, Supplementary Methods and Supplementary Results (PDF 2109 kb)

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Lee, S., Hong, S., Errey, J. et al. Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase. Nat Chem Biol 7, 631–638 (2011). https://doi.org/10.1038/nchembio.628

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