Séminaire Umberto

20/06/2024 - 11:30 - Salle Jacques Senez
Umberto CONTALDO, BIP 08

Deciphering the catalytic properties of the blue oxidase CueOs

Multicopper oxidases (MCOs) are enzymes that contain four Cu atoms spatially and functionally organized into two active sites. The mononuclear Cu-T1 performs phenol oxidation followed by electron transfer to the trinuclear copper center (TNC), which ultimately catalyzes the reduction of oxygen to water. CueOs belong to the MCO family and feature a methionine-rich (Met-rich) domain covering the Cu-T1. This results in lower phenol oxidase activity than in MCOs, (2), but enables CueO to catalyze the oxidation of cuprous to cupric ions, a crucial biological process. Interestingly, various structural organization and numbers of methionine residues within the Met-rich domain can be found depending on the microorganism (3)(4)(5). This raises the question of how Met-rich domains impact on CueOs’s cuprous oxidase activity, as well as bacterial copper resistance. We recently demonstrated that the Met-rich domain of EcCueO is dispensable for cuprous oxidation but is more likely a copper sensor domain (6). The structural organization of multiple solvent-accessible methionine residues facilitates the Cu+ recruitment from strongly chelated forms in vitro. With the aim of deciphering and validate the proposed role for the Met-rich domain, here we provide the biochemical, structural, spectroscopic, and catalytic characterization of a novel CueO from the Gram-negative bacteria Hafnia alvei (HaCueO). The interest in HaCueO is its high amino-acid conservation compared to EcCueO, the only difference being the composition of the Met-rich domain. We will show how a multimodal and multiscale approach allows to investigate the oxidative activities of HaCueO, ranging from in vitro to in vivo characterization. Our findings confirm the functional role of the Met-rich domain, allowing HaCueO to perform more efficiently cuprous oxidation compared to EcCueO.

(1) D. M. Mate, M. Alcalde, Laccase: a multi-purpose biocatalyst at the forefront of biotechnology. Microb. Biotechnol. 10, 1457–1467 (2017).
(2) K. Kataoka, et al., Structure and Function of the Engineered Multicopper Oxidase CueO from Escherichia coli-Deletion of the Methionine-Rich Helical Region Covering the Substrate-Binding Site. J. Mol. Biol. 373, 141–152 (2007).
(3) I. Mazurenko, et al., Electrochemistry of copper efflux oxidase-like multicopper oxidases involved in copper homeostasis. Curr. Opin. Electrochem. 32, 100919 (2022).
(4) V. P. Hitaishi, et al., Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction. JACS. 142, 1394–1405 (2020).
(5) R. Clément, et al., Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus. Biochimie 182, 228–237 (2021).
(6) U. Contaldo, et al., Methionine-rich domains emerge as facilitators of copper recruitment in detoxification systems. (under revision).



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