Mechanism of reconstitution/activation of the soluble PQQ-dependent glucose dehydrogenase from Acinetobacter calcoaceticus: a comprehensive study
| Autor: | Nicolas Mano, Benoît Limoges, Brice Kauffmann, Claire Stines-Chaumeil, François Mavré |
|---|---|
| Přispěvatelé: | Centre de Recherche Paul Pascal (CRPP), Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Electrochimie Moléculaire (LEM (UMR_7591)), Université Paris Diderot - Paris 7 (UPD7)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Européen de Chimie et Biologie (IECB), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherches Paul Pascal (CRPP), Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
General Chemical Engineering
[SDV]Life Sciences [q-bio] Protein Data Bank (RCSB PDB) 010402 general chemistry 01 natural sciences Article enzyme reconstitution 03 medical and health sciences chemistry.chemical_compound Pyrroloquinoline quinone Glucose dehydrogenase Enzyme reconstitution [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biochemistry [q-bio.BM] QD1-999 ComputingMilieux_MISCELLANEOUS 030304 developmental biology chemistry.chemical_classification glucose dehydrogenase 0303 health sciences biology Chemistry pyrroloquinoline quinone Biomolecules (q-bio.BM) General Chemistry [CHIM.CATA]Chemical Sciences/Catalysis enzyme cofactor biology.organism_classification Enzyme structure apoenzyme [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biomolecules [q-bio.BM] 0104 chemical sciences Enzyme binding Enzyme Quantitative Biology - Biomolecules FOS: Biological sciences Biophysics Acinetobacter calcoaceticus |
| Zdroj: | ACS Omega ACS Omega, ACS Publications, 2020, ⟨10.1021/acsomega.9b04034⟩ ACS Omega, Vol 5, Iss 4, Pp 2015-2026 (2020) |
| ISSN: | 2470-1343 |
| Popis: | The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e. the pyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated to two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamic, but also to demonstrate that when PQQ first binds to the apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca2+ binding loop through the specific mutation of the calcium coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB:5MIN). 45 pages, 11 figures, 6 tables |
| Databáze: | OpenAIRE |
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