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Mechanism of molybdate insertion into pterin-based molybdenum cofactors

Probst, Corinna and Yang, Jing and Krausze, Joern and Hercher, Thomas W. and Richers, Casseday P. and Spatzal, Thomas and KC, Khadanand and Giles, Logan J. and Rees, Douglas C. and Mendel, Ralf R. and Kirk, Martin L. and Kruse, Tobias (2021) Mechanism of molybdate insertion into pterin-based molybdenum cofactors. Nature Chemistry, 13 (8). pp. 758-765. ISSN 1755-4330. PMCID PMC8325642. https://resolver.caltech.edu/CaltechAUTHORS:20210628-180924312

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Abstract

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.


Item Type:Article
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URLURL TypeDescription
https://doi.org/10.1038/s41557-021-00714-1DOIArticle
https://rdcu.be/cnlZBPublisherFree ReadCube access
https://doi.org/10.2210/pdb6Q32/pdbRelated ItemData
ORCID:
AuthorORCID
Yang, Jing0000-0001-8241-9160
Krausze, Joern0000-0001-5333-8046
Richers, Casseday P.0000-0002-1324-0447
Spatzal, Thomas0000-0002-9136-5915
KC, Khadanand0000-0002-1258-8636
Rees, Douglas C.0000-0003-4073-1185
Mendel, Ralf R.0000-0003-3785-7764
Kirk, Martin L.0000-0002-1479-3318
Kruse, Tobias0000-0002-2716-3227
Additional Information:© The Author(s), under exclusive licence to Springer Nature Limited 2021. Received 29 April 2020. Accepted 27 April 2021. Published 28 June 2021. 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. Data availability. The protein structure data that support the findings of this study are publicly available from the Protein Data Bank (https://www.rcsb.org/) 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. These authors contributed equally: Corinna Probst, Jing Yang, Joern Krausze. These authors jointly supervised this work: Martin L. Kirk, Tobias Kruse. Author Contributions. 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. The authors declare no competing interests. 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.
Funders:
Funding AgencyGrant Number
NIHGM057378
NIHGM045162
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Department of Energy (DOE)DE-AC02-76SF00515
Deutsche Forschungsgemeinschaft (DFG)GRK 2223/1
Issue or Number:8
PubMed Central ID:PMC8325642
Record Number:CaltechAUTHORS:20210628-180924312
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210628-180924312
Official Citation:Probst, C., Yang, J., Krausze, J. et al. Mechanism of molybdate insertion into pterin-based molybdenum cofactors. Nat. Chem. 13, 758–765 (2021). https://doi.org/10.1038/s41557-021-00714-1
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109620
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:28 Jun 2021 21:49
Last Modified:26 Aug 2021 18:47

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