Identification of a robust bacterial pyranose oxidase that displays an unusual pH dependence.

Autor: Santema LL; Molecular Enzymology, University of Groningen, Groningen, The Netherlands., Rozeboom HJ; Molecular Enzymology, University of Groningen, Groningen, The Netherlands., Borger VP; Molecular Enzymology, University of Groningen, Groningen, The Netherlands., Kaya SG; Molecular Enzymology, University of Groningen, Groningen, The Netherlands., Fraaije MW; Molecular Enzymology, University of Groningen, Groningen, The Netherlands. Electronic address: m.w.fraaije@rug.nl.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2024 Nov; Vol. 300 (11), pp. 107885. Date of Electronic Publication: 2024 Oct 11.
DOI: 10.1016/j.jbc.2024.107885
Abstrakt: Pyranose oxidases are valuable biocatalysts, yet only a handful of bacterial pyranose oxidases are known. These bacterial enzymes exhibit noteworthy distinctions from their extensively characterized fungal counterparts, encompassing variations in substrate specificity and structural attributes. Herein a bacterial pyranose oxidase from Oscillatoria princeps (OPOx) was biochemically characterized in detail. In contrast to the fungal pyranose oxidases, OPOx could be well expressed in Escherichia coli as soluble, fully flavinylated, and active oxidase. It was found to be highly thermostable (melting temperature >90 °C) and showed activity on glucose, exhibiting an exceptionally low K M value (48 μM). Elucidation of its crystal structure revealed similarities with fungal pyranose oxidases, such as being a tetramer with a large central void leading to a narrow substrate access tunnel. In the active site, the FAD cofactor is covalently bound to a histidine. OPOx displays a relatively narrow pH optimum for activity with a sharp decline at relatively basic pH values which is accompanied by a drastic change in its flavin absorbance spectrum. The pH-dependent switch in flavin absorbance features and oxidase activity was shown to be fully reversible. It is hypothesized that a glutamic acid helps to stabilize the protonated form of the histidine that is tethered to the FAD. OPOx presents itself as a valuable biocatalyst as it is highly robust, well-expressed in E. coli, shows low K M values for monosaccharides, and has a peculiar pH-dependent "on-off switch".
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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