Structure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new membrane-associated domain and proton-relay system in catalysis

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dc.contributor.authorWójcik, Patrycja
dc.contributor.authorGlanowski, Michał
dc.contributor.authorMrugała, Beata
dc.contributor.authorProcner, Magdalena
dc.contributor.authorZastawny, Olga
dc.contributor.authorFlejszar, Monika
dc.contributor.authorKurpiewska, Katarzyna
dc.contributor.authorNiedziałkowska, Ewa
dc.contributor.authorMinor, Wladek
dc.contributor.authorOszajca, Maria
dc.contributor.authorBojarski, Andrzej
dc.contributor.authorWojtkiewicz, Agnieszka M.
dc.contributor.authorSzaleniec, Maciej
dc.contributor.organizationJerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciencesen
dc.contributor.organizationMaj Institute of Pharmacology Polish Academy of Sciencesen
dc.contributor.organizationDepartment of Physical Chemistry, Faculty of Chemistry, Rzeszow University of Technologyen
dc.contributor.organizationFaculty of Chemistry, Jagiellonian Universityen
dc.contributor.organizationDepartment of Molecular Physiology and Biological Physics, University of Virginiaen
dc.contributor.organizationMolecular Physiology and Biological Physics, University of Virginiaen
dc.date.accessioned2023-12-01T18:03:35Z
dc.date.available2023-12-01T18:03:35Z
dc.date.issued2023-01-10
dc.description.abstract3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and are useful find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid 1,2-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium substituted substrates. The role of the active site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. Finally, the modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ1-3- ketosteroids.en
dc.description.sponsorshipNarodowe Centrum Badań i Rozwoju: POWR. 03.02.00-00-I013/16 Narodowe Centrum Nauki: UMO-2016/21/B/ST4/03798
dc.identifier.citationBiochemistry 2023, 62, 3, 808–823. https://doi.org/10.1021/acs.biochem.2c00576en
dc.identifier.doi10.1021/acs.biochem.2c00576
dc.identifier.urihttps://open.icm.edu.pl/handle/123456789/23254
dc.language.isoen
dc.publisherAmerican Chemical Societyen
dc.rightsUznanie autorstwa 3.0 Polska*
dc.rights.urihttp://creativecommons.org/licenses/by/3.0/pl/*
dc.subjectketosteroid Δ1-dehydrogenaseen
dc.subject3-ketosteroidsen
dc.subjectΔ1-dehydrogenationen
dc.subject1(2)-dehydrogenationen
dc.subjectkinetic isotope effecten
dc.subject3-ketosteroid dehydrogenaseen
dc.titleStructure, mutagenesis and QM:MM modelling of 3-ketosteroid Δ1-dehydrogenase from Sterolibacterium denitrificans – the role of new membrane-associated domain and proton-relay system in catalysisen
dc.typearticle
dc.type.versionacceptedVersion
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