Browsing by Author "Urlacher, Vlada B."

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Copper-Radical Oxidases: A Diverse Group of Biocatalysts With Distinct Properties and a Broad Range of Biotechnological Applications
(Elsevier, 2022) Koschorreck, Katja; Alpdagtas, Saadet; Urlacher, Vlada B.
Copper-radical oxidases (CROs) catalyze the two-electron oxidation of a large number of primary alcohols including carbohydrates, polyols and benzylic alcohols as well as aldehydes and alpha-hydroxy-carbonyl compounds while reducing molecular oxygen to hydrogen peroxide. Initially, CROs like galactose oxidase and glyoxal oxidase were identified only in fungal secretomes. Since the last decade, their representatives have also been identified in some bacteria. CROs are grouped in the AA5 family of "auxiliary activities " in the database of Carbohydrate-Active enzymes. Despite low overall sequence similarity and different substrate specificities, sequence alignments and the solved crystal structures revealed a conserved architecture of the active sites in all CROs, with a mononuclear copper ion coordinated to an axial tyrosine, two histidines, and a cross-linked cysteine-tyrosyl radical cofactor. This unique post-translationally modified protein cofactor has attracted much attention in the past, which resulted in a large number of reports that shed light on key steps of the catalytic cycle and physico-chemical properties of CROs. Thanks to their broad substrate spectrum accompanied by the only need for molecular oxygen for catalysis, CROs since recently experience a renaissance and have been applied in various biocatalytic processes. This review provides an overview of the structural features, catalytic mechanism and substrates of CROs, presents an update on the engineering of these enzymes to improve their expression in recombinant hosts and to enhance their activity, and describes their potential fields of biotechnological application.
Identification of Redox Activators for Continuous Reactivation of Glyoxal Oxidase From Trametes Versicolor in a Two-Enzyme Reaction Cascade
(Nature Portfolio, 2024) Alpdagtas, Saadet; Jankowski, Nina; Urlacher, Vlada B.; Koschorreck, Katja
Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O-2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2 '-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.