Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mechanism of molybdate insertion into pterin-based molybdenum cofactors


The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here, we report this final maturation step, where adenylated MPT (MPT–AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco–AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT–AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: The four steps of Moco biosynthesis.
Fig. 2: Structure of Cnx1E variant S269D D274S with bound ligands.
Fig. 3: Moco–AMP dihedrals.
Fig. 4: Mo K-edge XAS spectra.
Fig. 5: Proposed reaction mechanism for Cnx1E-catalysed Mo insertion.

Data availability

The protein structure data that support the findings of this study are publicly available from the Protein Data Bank ( with accession code 6Q32. We deposited the protein structure along with the structure factor data file that allows for the re-computing and re-evaluation of the structure. Figures 2 and 3 and Supplementary Figs. 3, 4 and Extended Data Fig. 1 are associated with these raw data. Manuscript datasets are available as Supplementary Data files.


  1. Garner, C. D. et al. (eds) RSC Metallobiology Series Vols 1–3 (The Royal Society of Chemistry, 2019);

  2. Kirk, M. L. & Stein, B. in Comprehensive Inorganic Chemistry II 2dn edn (eds Reedijk, J. & Poeppelmeier, K.) 263–293 (Elsevier, 2013).

  3. Hille, R., Hall, J. & Basu, P. The mononuclear molybdenum enzymes. Chem. Rev. 114, 3963–4038 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Stiefel, E. L. The biogeochemistry of molybdenum and tungsten. Met. Ions Biol. Syst. 39, 1–29 (2002).

  5. Schwahn, B. C. et al. Efficacy and safety of cyclic pyranopterin monophosphate substitution in severe molybdenum cofactor deficiency type A: a prospective cohort study. Lancet 386, 1955–1963 (2015).

    CAS  PubMed  Google Scholar 

  6. Mendel, R. R. & Schwarz, G. Molybdenum cofactor biosynthesis in plants and humans. Coord. Chem. Rev. 255, 1145–1158 (2011).

    CAS  Google Scholar 

  7. Unkles, S. E. et al. The Aspergillus nidulans cnxABC locus is a single gene encoding two catalytic domains required for synthesis of precursor Z, an intermediate in molybdenum cofactor biosynthesis. J. Biol. Chem. 272, 28381–28390 (1997).

    CAS  PubMed  Google Scholar 

  8. Appleyard, M. et al. The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis—molecular characterization and analysis of in vivo generated mutans. J. Biol. Chem. 273, 14869–14876 (1998).

    CAS  PubMed  Google Scholar 

  9. Unkles, S. E., Heck, I. S., Appleyard, M. & Kinghorn, J. R. Eukaryotic molybdopterin synthase—biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants. J. Biol. Chem. 274, 19286–19293 (1999).

    CAS  PubMed  Google Scholar 

  10. Millar, L. et al. Deletion of the CnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans. Mol. Genet. Genomics 266, 445–453 (2001).

    CAS  PubMed  Google Scholar 

  11. Probst, C. et al. Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis. Fungal Genet. Biol. 66, 69–78 (2014).

    CAS  PubMed  Google Scholar 

  12. Mendel, R. R. The molybdenum cofactor. J. Biol. Chem. 288, 13165–13172 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Weiss, M. C. et al. The physiology and habitat of the last universal common ancestor. Nat. Microbiol 1, 116 (2016).

    Google Scholar 

  14. Hover, B. M., Loksztejn, A., Ribeiro, A. A. & Yokoyama, K. Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis. J. Am. Chem. Soc. 135, 7019–7032 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Hover, B. M., Tonthat, N. K., Schumacher, M. A. & Yokoyama, K. Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis. Proc. Natl Acad. Sci. USA 112, 6347–6352 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Wuebbens, M. M. & Rajagopalan, K. V. Structural characterization of a molybdopterin precursor. J. Biol. Chem. 268, 13493–13498 (1993).

    CAS  PubMed  Google Scholar 

  17. Teschner, J. et al. A novel role for Arabidopsis mitochondrial ABC transporter ATM3 in molybdenum cofactor biosynthesis. Plant Cell 22, 468–480 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Wuebbens, M. M. & Rajagopalan, K. V. Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis. J. Biol. Chem. 278, 14523–14532 (2003).

    CAS  PubMed  Google Scholar 

  19. Pitterle, D. M., Johnson, J. L. & Rajagopalan, K. V. In vitro synthesis of molybdopterin from precursor-Z using purified converting factor: role of protein-bound sulfur in formation of the dithiolene. J. Biol. Chem. 268, 13506–13509 (1993).

    CAS  PubMed  Google Scholar 

  20. Leimkuhler, S., Wuebbens, M. M. & Rajagopalan, K. V. The history of the discovery of the molybdenum cofactor and novel aspects of its biosynthesis in bacteria. Coord. Chem. Rev. 255, 1129–1144 (2011).

    PubMed  PubMed Central  Google Scholar 

  21. Kruse, T. Encyclopedia of Inorganic and Bioinorganic Chemistry (2020).

  22. Arst, H. N., MacDonald, D. W. & Cove, D. J. Molybdate metabolism in Aspergillus nidulans. Mutations affecting nitrate reductase and/or xanthine dehydrogenase. Mol. Gen. Genet. 108, 129–145 (1970).

    CAS  PubMed  Google Scholar 

  23. Joshi, M. S., Johnson, J. L. & Rajagopalan, K. V. Molybdenum cofactor biosynthesis in Escherichia coli mod and mog mutants. J. Bacteriol. 178, 4310–4312 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Schwarz, G. et al. The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton. Plant Cell 12, 2455–2471 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Llamas, A., Otte, T., Multhaup, G., Mendel, R. R. & Schwarz, G. The mechanism of nucleotide-assisted molybdenum insertion into molybdopterin—a novel route toward metal cofactor assembly. J. Biol. Chem. 281, 18343–18350 (2006).

    CAS  PubMed  Google Scholar 

  26. Kuper, J., Llamas, A., Hecht, H. J., Mendel, R. R. & Schwarz, G. Structure of the molybdopterin-bound Cnx1G domain links molybdenum and copper metabolism. Nature 430, 803–806 (2004).

    CAS  PubMed  Google Scholar 

  27. Llamas, A., Mendel, R. R. & Schwarz, N. Synthesis of adenylated molybdopterin—an essential step for molybdenum insertion. J. Biol. Chem. 279, 55241–55246 (2004).

    CAS  PubMed  Google Scholar 

  28. Krausze, J. et al. Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor. Biochem. J. 474, 163–178 (2017).

    CAS  PubMed  Google Scholar 

  29. Krausze, J. et al. The functional principle of eukaryotic molybdenum insertases. Biochem. J. 475, 1739–1753 (2018).

    CAS  PubMed  Google Scholar 

  30. Hercher, T. W. et al. Insights into the Cnx1E catalyzed MPT-AMP hydrolysis. Biosci. Rep. 40, BSR20191806 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Heck, I. S. et al. Mutational analysis of the gephyrin-related molybdenum cofactor biosyndietic gene cnxE from the lower eukaryote Aspergillus nidulans. Genetics 161, 623–632 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kirk, M. L. in Molybdenum and Tungsten Enzymes: Spectroscopic and Theoretical Investigations RSC Metallobiology Series No. 7 (eds Hille, R. et al.) 13–67 (The Royal Society of Chemistry, 2016).

  33. Schwarz, G., Boxer, D. H. & Mendel, R. R. in Chemistry and Biology of Pteridines and Folates (eds Pfleiderer, W. & Rokos, H.) 697–702 (Blackwell Science, 1997).

  34. Schwarz, G. & Mendel, R. R. Molybdenum cofactor biosynthesis and molybdoenzymes. Annu. Rev. Plant Biol. 57, 623–647 (2006).

    CAS  PubMed  Google Scholar 

  35. Rothery, R. A., Stein, B., Solomonson, M., Kirk, M. L. & Weiner, J. H. Pyranopterin conformation defines the function of molybdenum and tungsten enzymes. Proc. Natl Acad. Sci. USA 109, 14773–14778 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Xiang, S., Nichols, J., Rajagopalan, K. V. & Schindelin, H. The crystal structure of Escherichia coli MoeA and its relationship to the multifunctional protein gephyrin. Structure 9, 299–310 (2001).

    CAS  PubMed  Google Scholar 

  37. Kutzler, F. W. et al. Single-crystal polarized X-ray absorption spectroscopy. Observation and theory for (MoO2S2)2−. J. Am. Chem. Soc. 103, 6083–6088 (1981).

    CAS  Google Scholar 

  38. George, G. N. et al. Structure of the active site of sulfite oxidase. X-ray absorption spectroscopy of the molybdenum (iv), molybdenum (v) and molybdenum (vi) oxidation states. Biochemistry 28, 5075–5080 (1989).

    CAS  PubMed  Google Scholar 

  39. Partyka, D. V. & Holm, R. H. Oxygen/sulfur substitution reactions of tetraoxometalates effected by electrophilic carbon and silicon reagents. Inorg. Chem. 43, 8609–8616 (2004).

    CAS  PubMed  Google Scholar 

  40. Wang, J. J. & Holm, R. H. Silylation, sulfidation and benzene-1,2-dithiolate complexation reactions of oxo- and oxosulfidomolybdates(vi) and -tungstates(vi). Inorg. Chem. 46, 11156–11164 (2007).

    CAS  PubMed  Google Scholar 

  41. Lim, B. S., Willer, M. W., Miao, M. M. & Holm, R. H. Monodithiolene molybdenum(v,vi) complexes: a structural analogue of the oxidized active site of the sulfite oxidase enzyme family. J. Am. Chem. Soc. 123, 8343–8349 (2001).

    CAS  PubMed  Google Scholar 

  42. Harris, H. H., George, G. N. & Rajagopalan, K. V. High-resolution EXAFS of the active site of human sulfite oxidase: comparison with density functional theory and X-ray crystallographic results. Inorg. Chem. 45, 493–495 (2006).

    CAS  PubMed  Google Scholar 

  43. Liu, X. D., Cheng, J., Sprik, M. & Lu, X. C. Solution structures and acidity constants of molybdic acid. J. Phys. Chem. Lett. 4, 2926–2930 (2013).

    CAS  Google Scholar 

  44. George, G. N., Garrett, R. M., Prince, R. C. & Rajagopalan, K. V. The molybdenum site of sulfite oxidase: a comparison of wild-type and the cysteine 207 to serine mutant using X-ray absorption spectroscopy. J. Am. Chem. Soc. 118, 8588–8592 (1996).

    CAS  Google Scholar 

  45. Minubayeva, Z. & Seward, T. M. Molybdic acid ionisation under hydrothermal conditions to 300 °C. Geochim. Cosmochim. Acta 74, 4365–4374 (2010).

    CAS  Google Scholar 

  46. Schwarz, G., Boxer, D. H. & Mendel, R. R. Molybdenum cofactor biosynthesis—the plant protein Cnx1 binds molybdopterin with high affinity. J. Biol. Chem. 272, 26811–26814 (1997).

    CAS  PubMed  Google Scholar 

  47. Fischer, K. et al. Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii. J. Biol. Chem. 281, 30186–30194 (2006).

    CAS  PubMed  Google Scholar 

  48. Ringel, P. et al. Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa. J. Biol. Chem. 288, 14657–14671 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Kruse, T. et al. Identification and biochemical characterization of molybdenum cofactor-binding proteins from Arabidopsis thaliana. J. Biol. Chem. 285, 6623–6635 (2010).

    CAS  PubMed  Google Scholar 

  50. Stallmeyer, B., Nerlich, A., Schiemann, J., Brinkmann, H. & Mendel, R. R. Molybdenum cofactor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional 2-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins. Plant J. 8, 751–762 (1995).

    CAS  PubMed  Google Scholar 

  51. Leslie, A. G. W. & Powell, H. R. in Evolving Methods for Macromolecular Crystallography (eds Read, R. J. & Sussman, J. L.) 41–51 (Springer, 2007).

  52. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. STARANISO (Global Phasing Ltd, 2017).

  54. BUSTER v. 2.10.3 (Global Phasing Ltd, 2017).

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  57. The PyMOL Molecular Graphics System v. (Schrödinger, 2020).

  58. Pettersen, E. F. et al. UCSF chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Google Scholar 

  59. Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541 (2005).

    CAS  PubMed  Google Scholar 

  60. Frisch. M. J. et al. Gaussian 16, revision C.01 (Gaussian, Inc., 2016).

Download references


We thank A. Calean (TU Braunschweig) for excellent support with inductively coupled plasma mass spectrometry analysis. M.L.K. gratefully acknowledges support of this research by the National Institutes of Health (grant no. GM-057378). M.L.K. and J.Y. thank the UNM Center for Advanced Research Computing, supported in part by the National Science Foundation, for providing the computing resources used in this work. Work at Caltech was supported by the National Institutes of Health (NIH) grant no. GM045162. We acknowledge the Gordon and Betty Moore Foundation and the Beckman Institute at Caltech for their support of the Molecular Observatory at Caltech and the staff at beamlines 12-2 and 7-3. M.L.K. and J.Y. acknowledge the Stanford Synchrotron Radiation Lightsource, which is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. R.R.M. and T.K. acknowledge the support of this research by the Deutsche Forschungsgemeinschaft (GRK 2223/1). Correspondence and requests for materials should be addressed to M.L.K. and T.K.

Author information

Authors and Affiliations



C.P. carried out acquisition, analysis and interpretation of data. J.Y. carried out acquisition, analysis and interpretation of XAS data and computed the reaction coordinate. J.K. analysed and interpreted the data. T.W.H. carried out acquisition, analysis and interpretation of data. C.P.R. synthesized and characterized the trioxo- and dioxo-molybdenum model compounds (2, 3 and 4) and analysed spectroscopic data. T.S. carried out acquisition, analysis and interpretation of data. K.K. assisted with the collection of XAS data. L.J.G. assisted with the collection of XAS data. D.C.R. and R.R.M. revised the manuscript. M.L.K. conceived the idea, performed design analysis and interpretation of the data, and drafted the manuscript. T.K. conceived the idea and design, analysed and interpreted the data, and drafted the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Martin L. Kirk or Tobias Kruse.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Chemistry thanks Partha Basu, Carola Schulzke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The proposed mechanism of molybdate insertion into the MPT dithiolene moiety.

a, Ground state of Cnx1E with a Mg2+-water complex bound. Mg2+ constitutes the cofactor for the Cnx1E catalyzed reaction25,30. b, Conformation of MPT-AMP bound in the active site of Cnx1G26. c, First state of the catalysis with the substrate molybdate bound to the entry site. The presence of molybdate in the entry site induces an unfavorable (tense) backbone conformation in Cnx1E29. d, The conformation of MPT-AMP found in Cnx1G is not compatible with the Cnx1E active site28. Hence, the MPT-AMP pterin moiety must re-orient upon transfer from Cnx1G to Cnx1E, mostly involving two bond rotations. e, Second state of the catalytic cycle with molybdate bound to the entry site and active site bound MPT-AMP. The orientation of the pterin moiety was derived from active site bound Moco-AMP identified in this work. f, The transfer of molybdate from the entry to the insertion site is assisted by the relaxation of the tense backbone conformation29. According to our model of molybdate insertion, one molybdate oxygen ligand is two-fold protonated by the MPT dithiolene function and released as water. Simultaneously, the sulfur to molybdenum bonds are formed: This mechanism begins with a proton transfer from the diacid form of the MPT dithiolene side chain to a molybdate oxygen. A second, less activated protonation on the same oxygen atom would then yield a [(MPT)MoO3(OH2)]2- transition state. This [(MPT)MoO3(OH2)]2-species is expected to possess a highly labile aqua ligand, which would subsequently dissociate from Mo along the reaction coordinate to yield a more stable trioxo [(MPT)MoO3]2- intermediate. A final protonation of basic [(MPT)MoO3]2- by a suitable Cnx1E active site residue (most likely residue Arg369) yields our XAS derived [(MPT)MoO2(OH)]1- structure for Cnx1E Moco-AMP (for details see Fig. 5 and supplementary Fig. 11). The protonated oxygen atom is marked by an asterisk. g, The Cnx1E active site with bound Moco-AMP.

Supplementary information

Supplementary Information

Supplementary Figs 1–11 and Tables 1–5.

Supplementary Data 1

Primary data (dioxo (3)) Fig. 4a.

Supplementary Data 2

Primary data (insertase pH 6) Fig. 4a.

Supplementary Data 3

Primary data (insertase pH 8) Fig. 4a.

Supplementary Data 4

Primary data (molybdate) Fig. 4a.

Supplementary Data 5

Primary data (trioxo (2)) Fig. 4a.

Supplementary Data 6

Evaluated data Fig. 4b.

Supplementary Data 7

Primary data Fig.4c.

Supplementary Data 8

Primary data Fig. 4e.

Supplementary Data 9

Primary data Fig. 4f.

Supplementary Data 10

Primary data Supplementary Fig. 5a.

Supplementary Data 11

Primary data Supplementary Fig. 5b.

Supplementary Data 12

Primary data Supplementary Fig. 5c.

Supplementary Data 13

Primary HPLC data and evaluated data Supplementary Fig. 1a, replicate 1.

Supplementary Data 14

Calibration report replicate 1, Supplementary Fig. 1a.

Supplementary Data 15

Primary HPLC data and evaluated data Supplementary Fig. 1a, replicates 2 and 3.

Supplementary Data 16

Calibration report replicates 2 and 3, Supplementary Fig. 1a.

Supplementary Data 17

Primary ICP-MS Data and evaluated Data Suppl. Fig.1 A, Replicates 1–3.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Probst, C., Yang, J., Krausze, J. et al. Mechanism of molybdate insertion into pterin-based molybdenum cofactors. Nat. Chem. 13, 758–765 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing