Abstract
Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5′-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand β6 of the Pdx1 (βα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.
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Change history
23 January 2017
In the version of this article initially published online, reference to another recently published structure of the I320 complex was inadvertently omitted. The paper reporting the structure, which is now included as ref. 43 of the article, is Robinson, G.C., Kaufmann, M., Roux, C. & Fitzpatrick, T.B. Structural definition of the lysine swing in Arabidopsis thaliana PDX1: intermediate channeling facilitating vitamin B6 biosynthesis. Proc. Natl. Acad. Sci. USA 113, E5821–E5829 (2016). The error has been corrected in the print, PDF and HTML versions of this article.
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Acknowledgements
We thank W.J. Anderson, H. Clarke, C. Phippen (Southampton), A. Wessling and N. Kwak (Heidelberg), for their experimental contributions; T. Fitzpatrick (Geneva, Switzerland) for the generous gift of the expression plasmid for Arabidopsis Pdx1.3; S. Findlow and C. Holes at the Macromolecular Crystallisation Facility, Centre for Biological Sciences, and P. Horton and S. Coles at the Southampton Diffraction Centre, Chemistry, both University of Southampton; J. Kopp and C. Siegmann from the crystallization platform of the Cluster of Excellence CellNetworks, Heidelberg; staff at the Diamond Light Source and the European Synchrotron Radiation Facility for access and excellent user support without which this project would not have been possible; P. Carpentier, M. Weik, G. Gotthard and D. von Stetten at ESRF for support during data collection and online spectroscopy and D. Flot and G. Leonard for flexible access to the ESRF beamlines; A. Douangamath, P. Aller, R. Owen and M. Walsh for support at Diamond beamlines; B. Kappes (Erlangen) and P. Macheroux (Graz) for critical discussions; and L. Kinsland for assistance in preparation of the manuscript. This work was supported in parts by grants by the European Commission (VITBIOMAL-012158) and by the Deutsche Forschungsgemeinschaft (DFG) (TE368) to I.T., by the NIH (DK44083) and by the Robert A. Welch Foundation (A-0034) to T.P.B., by ESRF Mx1461, Mx1732, RADDAM and Diamond Light Source Mx8891 to I.T. M.J.R. was supported by a joint studentship between Diamond Light Source and the University of Southampton.
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M.J.R., V.W., G.G., S.W., M.S. and J.W.H. performed protein expression, purification and enzymatic essays. M.J.R., V.W., G.G., S.W., M.S. and I.T. performed crystallization and X-ray diffraction experiments. M.J.R., V.W., A.R. and I.T. performed online spectroscopy experiments. M.J.R., V.W., Y.Z., I.S., S.E.E., T.P.B. and I.T. performed crystallographic analysis and data deposition. M.J.R., A.R., G.E., T.P.B. and I.T. performed spectroscopic data analysis. M.J.R., G.E., I.S., S.E.E., T.P.B. and I.T. wrote the paper. M.J.R. and V.W. contributed equally to this work.
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Rodrigues, M., Windeisen, V., Zhang, Y. et al. Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis. Nat Chem Biol 13, 290–294 (2017). https://doi.org/10.1038/nchembio.2273
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DOI: https://doi.org/10.1038/nchembio.2273
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