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The active site of magnesium chelatase

Nature Plants volume 6, pages 1491–1502 (2020)Cite this article

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Abstract

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

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Fig. 1: The structure of the ChlH truncate T_ChlH_926.
Fig. 2: The structure of full-length Syn_ChlH.
Fig. 3: A model of PIX binding in Syn_ChlH.
Fig. 4: The E625K and E660W mutations alter the conformation of loops around the active site.
Fig. 5: A chain of buried solvent features are present adjacent to E660 in the Syn_ChlH structure.
Fig. 6: A proposed model for one-face sequential proton abstraction and magnesium ion insertion into PIX by ChlH.

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Data availability

The PDB accession codes are 6YS9, 6YSG, 6YT0, 6YTN and 6YTJ. The raw kinetic and biophysical data are available on request.

References

  1. Bryant, D. A., Hunter, C. N. & Warren, M. J. Biosynthesis of the modified tetrapyrroles—the pigments of life. J. Biol. Chem. https://doi.org/10.1074/jbc.REV120.006194 (2020).

  2. Medlock, A., Swartz, L., Dailey, T. A., Dailey, H. A. & Lanzilotta, W. N. Substrate interactions with human ferrochelatase. Proc. Natl Acad. Sci. USA 104, 1789–1793 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Medlock, A. E., Carter, M., Dailey, T. A., Dailey, H. A. & Lanzilotta, W. N. Product release rather than chelation determines metal specificity for ferrochelatase. J. Mol. Biol. 393, 308–319 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hoggins, M., Dailey, H. A., Hunter, C. N. & Reid, J. D. Direct measurement of metal ion chelation in the active site of human ferrochelatase. Biochemistry 46, 8121–8127 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Hofbauer, S., Helm, J., Obinger, C., Djinović-Carugo, K. & Furtmüller, P. G. Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase. FEBS J. https://doi.org/10.1111/febs.15164 (2020).

  6. Gillam, M. E., Hunter, G. A. & Ferreira, G. C. Ferrochelatase π-helix: implications from examining the role of the conserved π-helix glutamates in porphyrin metalation and product release. Arch. Biochem. Biophys. 644, 37–46 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gibson, L. C., Willows, R. D., Kannangara, C. G., von Wettstein, D. & Hunter, C. N. Magnesium–protoporphyrin chelatase of Rhodobacter sphaeroides: reconstitution of activity by combining the products of the BchH, -I, and -D genes expressed in Escherichia coli. Proc. Natl Acad. Sci. USA 92, 1941–1944 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Gibson, L. C., Jensen, P. E. & Hunter, C. N. Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI–BchD complex. Biochem. J. 337, 243–251 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Davison, P. A. et al. Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis. Biochemistry 44, 7603–7612 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Mochizuki, N., Brusslan, J. A., Larkin, R., Nagatani, A. & Chory, J. Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc. Natl Acad. Sci. USA 98, 2053–2058 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Larkin, R. M., Alonso, J. M., Ecker, J. R. & Chory, J. GUN4, a regulator of chlorophyll synthesis and intracellular signaling. Science 299, 902–906 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Jensen, P. E., Gibson, L. C., Henningsen, K. W. & Hunter, C. N. Expression of the chlI, chlD, and chlH genes from the Cyanobacterium synechocystis PCC6803 in Escherichia coli and demonstration that the three cognate proteins are required for magnesium-protoporphyrin chelatase activity. J. Biol. Chem. 271, 16662–16667 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Reid, J. D., Siebert, C. A., Bullough, P. A. & Hunter, C. N. The ATPase activity of the ChlI subunit of magnesium chelatase and formation of a heptameric AAA+ ring. Biochemistry 42, 6912–6920 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Reid, J. & Hunter, C. Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803. J. Biol. Chem. 279, 26893–26899 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Viney, J., Davison, P. A., Hunter, C. N. & Reid, J. D. Direct measurement of metal-ion chelation in the active site of the AAA+ATPase magnesium chelatase. Biochemistry 46, 12788–12794 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Adams, N. B. P. & Reid, J. D. The allosteric role of the AAA+ domain of ChlD protein from the magnesium chelatase of Synechocystis species PCC 6803. J. Biol. Chem. 288, 28727–28732 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Adams, N. B. P. et al. Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus. Biochem. J. 464, 315–322 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Brindley, A. A., Adams, N. B. P., Hunter, C. N. & Reid, J. D. Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg2+ cooperativity upon magnesium chelatase. Biochemistry 54, 6659–6662 (2015).

    Article  CAS  PubMed  Google Scholar 

  19. Adams, N. B. P., Brindley, A. A., Hunter, C. N. & Reid, J. D. The catalytic power of magnesium chelatase: a benchmark for the AAA+ATPases. FEBS Lett. 590, 1687–1693 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Farmer, D. A. et al. The ChlD subunit links the motor and porphyrin binding subunits of magnesium chelatase. Biochem. J. 476, 1875–1887 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Reid, J. D. & Hunter, C. N. Current understanding of the function of magnesium chelatase. Biochem. Soc. Trans. 30, 643–645 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Qian, P. et al. Structure of the cyanobacterial magnesium chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering. J. Biol. Chem. 287, 4946–4956 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Chen, X. et al. Crystal structure of the catalytic subunit of magnesium chelatase. Nat. Plants 1, 15125 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Chen, Z., Zhang, X., Liu, Y. & Jiang, L. Crystal structure of the Arabidopsis thaliana C-terminal ChlH at 1.25 Å (PDB, 2016); https://doi.org/10.2210/pdb5ewu/pdb

  25. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Karger, G. A., Reid, J. D. & Hunter, C. N. Characterization of the binding of deuteroporphyrin IX to the magnesium chelatase H subunit and spectroscopic properties of the complex. Biochemistry 40, 9291–9299 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Porter, C. M. & Miller, B. G. Cooperativity in monomeric enzymes with single ligand-binding sites. Bioorg. Chem. 43, 44–50 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Adams, N. B. P., Marklew, C. J., Brindley, A. A., Hunter, C. N. & Reid, J. D. Characterization of the magnesium chelatase from Thermosynechococcus elongatus. Biochem. J. 457, 163–170 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Cornish-Bowden, A. Fundamentals of Enzyme Kinetics (Portland Press, 2004).

  30. Schubert, H. L., Raux, E., Wilson, K. S. & Warren, M. J. Common chelatase design in the branched tetrapyrrole pathways of heme and anaerobic cobalamin synthesis. Biochemistry 38, 10660–10669 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Al-Karadaghi, S., Hansson, M., Nikonov, S., Jönsson, B. & Hederstedt, L. Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis. Structure 5, 1501–1510 (1997).

    Article  CAS  PubMed  Google Scholar 

  32. Wu, C.-K. et al. The 2.0 A structure of human ferrochelatase, the terminal enzyme of heme biosynthesis. Nat. Struct. Biol. 8, 156–160 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Karlberg, T. et al. Metal binding to Saccharomyces cerevisiae ferrochelatase. Biochemistry 41, 13499–13506 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Dailey, H. A. et al. Ferrochelatase at the millennium: structures,mechanisms and [2Fe-2S] clusters. Cell.Mol. Life Sci. 57, 1909–1926 (2020).

    Article  Google Scholar 

  35. Dailey, H. A. et al. Prokaryotic heme biosynthesis: multiple pathways to a common essential product. Microbiol. Mol. Biol. Rev. https://doi.org/10.1128/MMBR.00048-16 (2017).

  36. Shen, Y. & Ryde, U. Reaction mechanism of porphyrin metallation studied by theoretical methods. Chemistry 11, 1549–1564 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Chen, G. E. et al. Complete enzyme set for chlorophyll biosynthesis in Escherichia coli. Sci. Adv. https://doi.org/10.1126/sciadv.aaq1407 (2018).

  38. Smith, K. M. & Goff, D. A. Syntheses of some proposed biosynthetic precursors to the isocyclic ring in chlorophyll a. J. Org. Chem. 51, 657–666 (1986).

    Article  CAS  Google Scholar 

  39. Jensen, P. E., Gibson, L. C. D. & Hunter, C. N. Determinants of catalytic activity with the use of purified I, D and H subunits of the magnesium protoporphyrin IX chelatase from Synechocystis PCC6803. Biochem. J. 334, 335–344 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sawicki, A. & Willows, R. D. Kinetic analyses of the magnesium chelatase provide insights into the mechanism, structure, and formation of the complex. J. Biol. Chem. 283, 31294–31302 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Celis, A. I. & DuBois, J. L. Making and breaking heme. Curr. Opin. Struct. Biol. 59, 19–28 (2019).

    Article  CAS  PubMed  Google Scholar 

  42. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  43. Sheldrick, G. M. & Schneider, T. R. in Methods in Enzymology: Macromolecular Crystallography Part B Vol. 277, 319–343 (Academic Press, 1997); https://doi.org/10.1016/S0076-6879(97)77018-6

  44. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006).

    Article  PubMed  CAS  Google Scholar 

  46. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  47. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  48. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Nicholls, R. A., Long, F. & Murshudov, G. N. Low-resolution refinement tools in REFMAC5. Acta Crystallogr. D 68, 404–417 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Nicholls, R.A., Kovalevskiy, O. & Murshudov, G. N. In Protein Crystallography. Methods in Molecular Biology Vol. 1607 (eds Wlodawer A et al.) 565–593 (Humana Press, 2017).

  51. O’Boyle, N. M., Banck, M., James, C. A. et al. Open Babel: An open chemical toolbox. J. Cheminform. 3, 33 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  52. Forli, S. et al. Computational protein–ligand docking and virtual drug screening with the AutoDock suite. Nat. Protoc. 11, 905–919 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Walser, M. Ion association v. dissociation constants for complexes of citrate with sodium, potassium, calcium, and magnesium ions. J. Phys. Chem. 65, 159–161 (1961).

    Article  CAS  Google Scholar 

  54. Meneely, K. M., Sundlov, J. A., Gulick, A. M., Moran, G. R. & Lamb, A. L. An open and shut case: the interaction of magnesium with MST enzymes. J. Am. Chem. Soc. 138, 9277–9293 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Stein, R. L. Kinetics of Enzyme Action (John Wiley, 2011).

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Acknowledgements

We thank Diamond Light Source for beamtime (proposals mx8987 and 12788) and the staff of beamlines i24, i02, i03 and i04 for technical support. We thank D. Ladakis, R. W. Pickersgill, D. G. Brown and M. J. Warren for the provision of structural data on the truncation of the CobN protein. N.B.P.A., C.B., A.A.B., P.A.D., J.D.R. and C.N.H. acknowledge financial support from the Biotechnology and Biological Sciences Research Council (BBSRC UK) (award no. BB/M000265/1). C.B. was also supported by core funding from the Department of Molecular Biology and Biotechnology, University of Sheffield. C.N.H was also supported by Synergy Award no. 854126 from the European Research Council. D.A.F. was supported by a BBSRC White Rose Doctoral Training Program (award no. BB/M011151/1).

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Author notes

  1. Claudine Bisson

    Present address: Centre for Ultrastructural Imaging, New Hunt’s House, Guy’s Campus, King’s College London, London, UK

  2. These authors contributed equally: Nathan B. P. Adams, Claudine Bisson.

Authors and Affiliations

  1. Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK

    Nathan B. P. Adams, Claudine Bisson, Amanda A. Brindley, David A. Farmer, Paul A. Davison & C. Neil Hunter

  2. Department of Chemistry, The University of Sheffield, Sheffield, UK

    James D. Reid

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Contributions

N.B.P.A. and D.A.F. carried out the kinetics and biophysics experiments. A.A.B. and P.A.D. generated the mutants and with N.B.P.A. and D.A.F. prepared the proteins. A.A.B., N.B.P.A. and C.B. set up the crystallization experiments. C.B. determined, built and analysed the structures. C.N.H. provided laboratory space and materials to carry out the work. N.B.P.A., C.B. and C.N.H. wrote the manuscript with contributions from J.D.R. All authors approved the manuscript.

Corresponding authors

Correspondence to Nathan B. P. Adams or C. Neil Hunter.

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Peer review information Nature Plants thanks Salam Al-Karadaghi, Robert Willows and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data

Extended Data Fig. 1 The magnesium chelatase reaction.

Magnesium chelatase (ChlIDH) catalyses the ATP dependent conversion of protoporphyrin IX (PIX) into magnesium protoporphyrin IX (MgPIX). The pyrrole rings are identified by the letter in blue.

Extended Data Fig. 2 Data collection and refinement statistics.

a Rmerge = ∑hkl∑i∣Ii − Im∣/∑hkl∑iIi, \({}^{{\rm{b}}}\ {R}_{pim}={\sum }_{{\rm{hkl}}}\sqrt{1}/n-1{\sum }_{i = 1}| {I}_{i}-{I}_{m}| /{\sum }_{{\rm{hkl}}}{\sum }_{i}{I}_{i}\), where Ii and Im are the observed intensity and mean intensity of related reflections, respectively. c Values in parenthesis are for data in the high-resolution shell.

Extended Data Fig. 3 Fit coefficients for mutations in the active site of the ChlH protein with respect to [DIX].

Data from Fig. 3 panels d, and e, is described by equation 5 unless stated otherwise, errors reported as one standard deviation of the fit coefficient. n.d. no activity detected. * these residues are adjacent to site 1. † Fitted to the Hill equation (6), where s0.5 is reported instead of KM, and kcat/s0.5 instead of kcat/KM. Fitted to the substrate inhibition equation (7), where Ki = 37.1 ± 7.7 μM.

Extended Data Fig. 4 Characterisation of the E660D and H1174V mutants.

a, and c, DIX and b, and d, Mg2+ dependence of the steady state rate of Mg2+ chelation for WT ChlH (closed circles), H1174V (open circles), E660D (open squares). Assays contained 0.1 mM ChlD, 0.2 mM ChlI, 0.4 mM WT ChlH or H1174V in 50 mM MOPS/KOH, 0.3 M glycerol, 1 mM DTT, 10 mM free Mg2+, 5 mM MgATP2− unless stated otherwise. Lines are theoretical, where steady state rates (vss) were fitted to the Michaelis-Menten equation (Equation (5)) in panel a and c or the Hill equation (Equation (6)) in panel b and d, and kinetic coefficients reported in Extended Data Table 3. Each data point is an individual experiment.

Extended Data Fig. 5 Characteristic steady state parameters of WT ChlH, H1174V and E660D proteins.

Data from Extended Data Fig. 2. When magnesium is the varied substrate, data is fitted to Equation (6): * S0.5, † kcat/S0.5. Values reported are the errors reported as one standard deviation of the fit coefficient.

Extended Data Fig. 6 Energy transfer from tryptophans to porphyrin in the active site.

a, The structure of ChlH represented as black ribbon with all native tryptophan residues represented as black spheres. The control mutation, E263, is shown as cyan spheres, as is the active site E660, with PIX docked in the active site represented as green spheres. b, Fluorescence emission of DIX after excitation or tryptophans at 280 nM. Black, WT ChlH; blue, E263W, Red, E660W. Averages of three independent biological repeats are represented with a central solid line and shading representing standard deviation. The peak areas in the insert are the total integrated number of counts for the full wavelength range shown in the graph. The bars represent the mean value from three independent experiments, with error bars ± the standard deviation. In addition, each area value is plotted for as an open circle.

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Adams, N.B.P., Bisson, C., Brindley, A.A. et al. The active site of magnesium chelatase. Nat. Plants 6, 1491–1502 (2020). https://doi.org/10.1038/s41477-020-00806-9

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