2026 FrenchBIC post-doc prize to Umberto Contaldo

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During my postdoctoral research at the Bioénergétique et Ingénierie des Protéines laboratory (BIP) in the team of Elisabeth Lojou, I investigated the molecular mechanisms underlying bacterial copper homeostasis through the study of a multicopper oxidase called CueO. Copper is an essential yet potentially toxic element for living organisms, and maintaining its intracellular balance is critical for bacterial survival, particularly in the context of host-pathogen interactions. In Gram-negative bacteria such as Escherichia coli, copper homeostasis relies on a finely tuned network of transporters and enzymes that ensure both sufficient supply for metalloprotein biosynthesis and protection against toxic accumulation. Within this system, CueO plays a key role by catalyzing the oxidation of the highly reactive cuprous ion (Cu+) into the less toxic cupric form (Cu2+), thereby contributing to cellular detoxification while limiting the formation of harmful reactive oxygen species (Figure A).

CueO belongs to the multicopper oxidase family, a superfamily of enzymes characterized by a conserved fold hosting four copper atoms organized into distinct functional centers. A striking feature of bacterial CueO enzymes is the presence of a flexible insertion enriched in histidine and methionine residues (His/Met-rich), whose precise functional role has long remained unclear. This domain is capable of binding copper ions, yet its contribution to catalysis and copper handling has been debated (Figure B).

My work aimed to clarify the role of this His/Met-rich insertion and to identify the molecular determinants governing Cu+ oxidation and enzyme function. To address this, I developed a multiscale and multimodal strategy combining in vivo detoxification assays with in vitro catalytic and electrocatalytic analyses (Contaldo et al, PNAS 2024). This approach enabled me to disentangle the respective contributions of different structural elements of the enzyme. I demonstrated that the His/Met-rich insertion is not strictly required for Cu+ oxidation under conditions of high copper availability. Instead, I identified a specific copper-binding site, termed Cu5, located in close proximity to the primary electron-transfer center, as the key locus for efficient catalytic oxidation (Figure C). I further showed that the integrity of this site is essential for enzyme activity both in vitro and in vivo.

Figure A) Schematic representation of the major copper homeostasis systems in Escherichia coli. B) Superimposed crystal structures of E. coli CueO: PDB 3OD3 showing the full-length and Cu-T1/TNC, and PDB 3NT0 highlighting the auxiliary copper-binding sites Cu5, Cu6, Cu7 and Cu8. The protein backbone is shown as a ribbon diagram scaled by B-factor (min 0.1 Å, max 2.0 Å) with the His/Met-rich insertion highlighted in lime green. Copper atoms are depicted as spheres with van der Waals radii of 2.0 Å, and coordinating residues are shown as sticks. C) Proposed cuprous oxidase reaction mechanisms of EcCueO. D) Alignment of His/Met-rich insertion sequences from EcCueO and HaCueO. Methionine and histidine residues are highlighted in yellow and red, respectively. EcCueO contains 13 methionine residues, whereas HaCueO contains 19; both enzymes contain 7 histidine residues.

I then extended this work by investigating the diversity of CueO homologs across bacterial species. These enzymes display significant variability in the length and composition of their His/Met-rich insertions, suggesting an adaptive role. To explore this hypothesis, I performed a comparative study between CueO from Escherichia coli and a homolog from Hafnia alvei, combining protein engineering, structural biology, and functional characterization (Figure D) (Contaldo et al, JACS Au 2025). By generating and analyzing chimeric variants in which the flexible His/Met-rich insertion was exchanged between the two enzymes, I demonstrated that the size and composition of this domain directly modulate catalytic properties (Saska et al, Bioelectrochemistry 2025). In particular, larger insertions enhance copper acquisition under conditions where the metal is strongly chelated, likely by promoting transient binding and efficient substrate recruitment. Conversely, these extended domains can also introduce steric constraints that limit access of certain substrates to the catalytic site, highlighting a trade-off between accessibility and affinity.

More recently, I characterized an additional transient copper-binding site, termed Cu8, which plays a crucial role in the metalation of the enzyme (Figure B) (Santucci et al, JIB 2026). I showed that this site is required for efficient incorporation of copper into the protein under conditions of low metal availability, suggesting that it may serve as an interaction interface with periplasmic copper chaperones in vivo. This finding provides new insight into the maturation process of multicopper oxidases and reveals an additional layer of regulation in bacterial copper homeostasis.

Altogether, this work provides a comprehensive understanding of how structural features within CueO enzymes govern their catalytic function and adaptation to environmental conditions. By combining mechanistic, structural, and comparative approaches, I uncovered key principles linking protein architecture to copper handling in bacteria. These findings not only advance fundamental knowledge of metalloprotein function and bacterial physiology but also open perspectives for the development of biotechnological applications. In particular, the ability of CueO enzymes to oxidize strongly chelated copper suggests promising potential for their use as biosensors or detoxification tools in complex and metal-contaminated environments.

  • U. Contaldo, D. Savant-Aira, A. Vergnes, J. Becam, F. Biaso, M. Ilbert, L. Aussel, B. Ezraty, E. Lojou, I. Mazurenko, Methionine-rich domains emerge as facilitators of copper recruitment in detoxification systems, Proc. Natl. Acad. Sci. 121 (2024) e2402862121. 10.1073/pnas.2402862121
  • U. Contaldo, P. Santucci, A. Vergnes, P. Leone, J. Becam, F. Biaso, M. Ilbert, B. Ezraty, E. Lojou, I. Mazurenko, How the Larger Methionine-Rich Domain of CueO from Hafnia alvei Enhances Cuprous Oxidation, JACS Au 5 (2025) 1833–1844. 10.1021/jacsau.5c00076
  • V. Saska, P. Santucci, A. de Poulpiquet, D. Gasparutto, U. Contaldo, I. Mazurenko, E. Lojou, Tuning O2 enzymatic reduction: Roles of methionine-rich domains and electrochemical metalation of active centers, Bioelectrochemistry 166 (2025) 109051 10.1016/j.bioelechem.2025.109051
  • P. Santucci, F. Biaso, J. Becam, L. Dubard, M. Ilbert, B. Ezraty, I. Mazurenko, E. Lojou, U. Contaldo, A conserved copper-binding site in multicopper oxidases regulates the metalation of CueO from Escherichia coli, J. Inorg. Biochem. 278 (2026). 10.1016/j.jinorgbio.2026.113257

Current affiliation: Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, Aix Marseille Université, Marseille, France.

Past affiliations: PhD thesis: Christine Cavazza & Alan Le Goff, French Atomic Energy and Alternative Energy Commission (CEA) & Department of Molecular Chemistry, University of Grenoble Alpes. Master internship/fellowship: S. Ciurli laboratory, Department of Pharmacy and Biotechnology, Alma Mater Studiorum, University of Bologna.


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