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

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  • The ability of flavoenzymes to reduce dioxygen varies greatly and is controlled by the protein environment that can cause either a rapid (oxidases) or sluggish (dehydrogenases) reaction. 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 alternate gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily, which has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternate 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) lowered its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes have a cavity binding an anion and resembling 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 might be governed by a common mechanistic principle.
  • This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by FEBS (Federation of European Biochemical Societies) and published by John Wiley & Sons, Inc. It can be found at: http://febs.onlinelibrary.wiley.com/hub/journal/10.1111/%28ISSN%291742-4658/
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  • Zafred, D., Steiner, B., Teufelberger, A. R., Hromic, A., Karplus, P. A., Schofield, C. J., Wallner, S., & Macheroux, P. (2015). Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction. FEBS Journal, 282(16): 3060–3074. doi:10.1111/febs.13212
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  • 282
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  • 16
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  • This research was supported by the “Fonds zur Förderung der wissenschaftlichen Forschung” (FWF) through SFB projects F1805 and F4604 and by the PhD program “Molecular Enzymology” (W901). A short-term visit of D. Zafred to the laboratory of Prof. C. J. Schofield was supported by EMBO. We are grateful to Prof. Walter Keller for his financial support and resourceful discussions.
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