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Metal uptake and metalloproteins

Published on 26 April 2019

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Pascal ARNOUX, Mila KOJADINOVIC, Monique SABATY & David PIGNOL​​

Technician/Ingin​eer : Sandrine GROSSE,& Catherine BRUTESCO

PhD Student:: Pierre-Xavier MAZIANI

Posdoc: Lionel TARRAGO

This topics is dedicated to the study of molecular mechanisms responsible for metal uptake and metalloproteins maturation. Genetics coupled with structural biochemistry approaches are applied to obtain structural informations on metallo-enzyme families poorly characterized (Molybdoenzymes), and on original enzymatic synthesis of metal chelators (metallophores). These approaches potentially contributes to the discovery of original catalytic mechanisms as well as new compounds with unknown properties. This fundamental axis is also feeding biotechnological approaches, with developments in bioremediation and biosensing.

Molybdo-enzymes

Two Molybdo-enzymes from Rhodobacter sphaeroides Rb are characterized: (i) the periplasmic nitrate reductase (NapAB) which can reduce nitrate but also selenium oxide, and (ii) MsrP, an oxidoreductase playing a role in the repair of oxidative stress induced by the metallic stress. These metalloproteins are studied at the structural (1), biochemical (2,3, 15) and biophysical (4, 5, 6, 7, 8, 9, 14) levels. A project funded by the National Research Agency (ANR MC2, MOLYERE and METOXIC) aims at  understanding the substrate specificity of these metalloproteins.​

Metal chelators 

Metals are required in almost every life processes and therefore all life forms have developed some mechanisms of metal acquisition​. We focusse on the characterization of orignial metal uptake in bacteria.

Nicotianamine (NA) is a small molecule present in all plants. NA binds various metal ions such as Cu2+ Zn2+ Mn2+ and Ni2+. Nicotianamine synthase (NAS) catalyzes the biosynthesis of NA by condensation of three molecules of S-adenosylmethionine. Usinga structural approach on an archeal enzyme related to eukaryotic NAS, we were able to describe for the first time a unique enzymatic mechanism responsible for the synthesis of NA (10,11,12). Similar study on NAS-like from bacteria is currently underway.

Staphylopine: Characterization of the nicotianamine synthase (NAS) family, together with an extensive genomic analysis allowed us to propose the existence of NAS-Like enzymes in the bacterial domain. Studying this system through metabolomic exploration, targeted mutagenesis, and biochemical analysis, we uncovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking (export and import systems) of a broad-spectrum NA-like metallophore (coined staphylopine). Biosynthesis of staphylopine requires the association of three unprecedented enzyme activities. We further demonstrated that staphylopine is involved in nickel, cobalt, zinc, copper and iron acquisition, depending on the growth conditions. Because this biosynthetic pathway is conserved in several pathogens our work underscores the importance of this metal acquisition strategy in bacterial infection. This discovery was recently accepted for publication in the journal Science (18) and paves the way to numerous potential biomedical applications .​​


















structure of the periplasmic nitrate reductase (NapAB) 
 

X-ray structure of NAS from Methanothermobacter thermautotrophicus
 
 
Model of staphylopine function, involving three biosynthetic enzymes (blue, gray and fuchsia) that use common precursors, one exporter (magenta), and an importer belonging to the family of ABC transporters (simplified here in orange
Crédit : Pascal Arnoux/CEA
Collaborators
  • A. Magalon and F. Barras (LCB Marseille)
  • R. Voulhoux (LISM, Marseille)
  • E. Borezée-Durant (INRA, Jouy en Josas)
  • R.​ Lobinski & L. Ouerdane (Université de Pau et du Pays de l'Adour)
  • M. Carrière (CEA Grenoble)
Fundings
  • ANR MC2 (2011-2015), coordinator C. Leger (BIP Marseille)
  • Programme Toxnuc ( BAMACO), coordinator D. Pig​nol
  • ANR ANIBAL (2015-2018), coordinator P. Arnoux.
  • ANR METOXIC (2016-2019), coordinator B. Ezrati (LCB, Marseille)
  • VLM (2016-2019), coordinator R Voulhoux (LISM, Marseille).
Publications​

 

  1. Arnoux P, Sabaty M, Alric J, Frangioni B, Guigliarelli B, Adriano JM, Pignol D (2003) Structural and redox plasticity in the heterodimeric periplasmic nitrate reductase.Nature Struct. Biol. 10(11):928-934. 
  2. Dementin S, Arnoux P, Frangioni B, Grosse S, Léger C, Guigliareli B, Burlat B, Sabaty M, Pignol D (2007) Insights into the substrate binding site of the periplasmic nitrate reductase from site direct mutagenesis.Biochemistry 46(34):9713-9721.
  3. Pierru B, Grosse S, Pignol D, Sabaty M (2006) Genetic and biochemical evidences for the 1 involvement of a molybdo-enzyme in one of the selenite reduction pathways of Rhodobacter sphaeroides f. sp. denitrificans IL106.Appl Env. Microb.72(5):3147-3153.
  4. Bertrand P, Frangioni B, Dementin S, Guigliarelli B, Sabaty M, Arnoux P, Pignol D, Léger C (2007) Effects of slow substrate binding & release in redox enzymes: theory and application to periplasmic nitrate reductase.J. Phys. Chem. 111(34):10300-1031.
  5.  Fourmond V, Sabaty M, Arnoux P, Bertrand P, Pignol D, Léger C (2010) Reassessing the strategies for trapping catalytic intermediates during nitrate réductase turn-over. J Phys Chem B 114(9):3341-3347.
  6. Fourmond V, Burlat B, Dementin S, Sabaty M, Arnoux P, Etienne E, Guigliarelli B, Bertrand P, Pignol D, Leger C (2010) Dependence of catalytic activity on driving force in solution assays and protein film voltammetry. Biochemistry-US 49(11):2424-
  7. Fourmond V, Lautier T, Baffert C, Leroux F, Dementin S, Liebogg P, Rousset M, Arnoux P, Pignol D, Meynial Salles I, Soucaille P, Bertrand P, Léger C (2009) Correcting for electrocatalyst dersorbtion or inactivation in chronoamperometry experiments.Anal Chem, 15;81(8):2962-2968 doi: 10.1021/ac8025702.
  8.  Fourmond V, Burlat B, Dementin S, Arnoux P, Sabaty M, Boiry S, Guigliarelli B, Bertrand P, Pignol D, Léger C. (2008) J Phys Chem B.Major Mo(V) EPR signature of Rhodobacter sphaeroides periplasmic nitrate reductase arising from a dead-end species that activates upon reduction. Relation to other molybdoenzymes from the DMSO reductase family. 112(48):15478-15486. doi: 10.1021/jp807092y.
  9. Frangioni B, Arnoux P, Pignol D, Sabaty M, Bertrand P, Guigliarelli B (2004) In Rhodobacter sphaeroides respiratory nitrate reductase, the kinetics of substrate binding disrupts the energetics of the catalytic cycle and favors intramolecular electron transfer. J. Am. Chem. Soc. Feb 11;126(5):1328-1329. 
  10. Dreyfus C, Pignol D, Arnoux P (2008) Expression, purification, cristallization and preliminary X-ray analysis of an archael protein homologous to plant nicotianamine synthase. Acta Cryst. F64, 933-935 
  11.  Dreyfus C, Lemaire D, Mari S, Pignol D, Arnoux P (2009) Crystallographic snapshots of substrate translocation during phytosiderophore synthesis. Proc. Natl. Acad. Sci. U S A. 106(38):16180-16184. 
  12.  Dreyfus C, Larrouy M, Cavelier F, Martinez J, Pignol D, Arnoux P (2011) Crystallographic structure of thermoNicotianamine synthase with a synthetic reaction intermediate highlights the sequential processing mechanism. Chem Commun (Camb). 28;47(20):5825.
  13.  Vivares D, Arnoux P, Pignol D (2005) A papain-like enzyme at work : crystal structure of native and acyl-enzyme intermediates in phytochelatin synthesis. Proc. Natl. Acad. Sci. U S A. 102(52):18848–18853.
  14. Jacques J, Fourmond V,Arnoux P,Sabaty M, Emilien E, Grosse S, Biaso F, Bertrand P, Pignol D, Léger C, Guigliarelli B, Burlat B (2014) Reductive activation in periplasmic nitrate reductase involves chemical modifications of the Mo-cofactor beyond the first coordination sphere of the Mo ion.Biochim Biophys Acta.doi: 10.1016/j.bbabio.2013.10.013.
  15. Sabaty M, Grosse S, Adryanczyk G, Boiry S, Biaso F, Arnoux P, Pignol D (2013) Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis.BMC Biochem. 2013 Nov 1;14(1):28
  16. Jacques J, Burlat B, Arnoux P, Sabaty M, Guigliarelli B, Léger C, Pignol D, Fourmond V. (2014) Kinetics of substrate inhibition of periplasmic nitrate reductase. BBA – Bioenergetics doi: 10.1016/j.bbabio.2014.05.357
  17.  Arnoux P., Ruppelt C., Oudouhou F., Lavergne J., Siponen M.I., Toci R.,  Bittner F., Mendel R.R., Pignol D., Magalon A. & Walburger A. (2015) "Sulfur shuttling across a chaperone during molybdenum cofactor maturation"  Nature Communication  2015 Feb 4;6:6148. doi: 10.1038/ncomms7148.

  18. Ghssein G, Brutesco C, Ouerdane L, Fojcik C, Izaute A, Hajjar C, Lobinski R, Lemaire D, Richaud P, Voulhoux R, Espaillat A, Cava F, Pignol D, Borezee-Durant E & Arnoux P (2016) Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus. Science    May 27;352(6289):1105-9. doi: 10.1126/science.aaf1018.​    

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