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Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction

DOI: 10.1111/febs.13212 DOI Help
PMID: 25619330 PMID Help

Authors: Domen Zafred (University of Graz) , Barbara Steiner (University of Graz) , Andrea R. Teufelberger (University of Graz) , Altijana Hromic (University of Graz) , P. Andrew Karplus (Oregon State University) , Christopher J. Schofield (University of Oxford) , Silvia Wallner (Graz University of Technology) , Peter Macheroux (Graz University of Technology)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Febs Journal , VOL 282 (16) , PAGES 3060 - 3074

State: Published (Approved)
Published: August 2015

Abstract: The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a ‘gatekeeper’ amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60 000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the ‘oxyanion hole’ in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle.

Journal Keywords: Dehydrogenase; Enzyme Design; Oxidase; Oxyanion Hole; Oxygen Reactivity

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)