Rationally engineered flavin‐dependent oxidase reveals steric control of dioxygen reduction. (24th February 2015)

• Record Type: Journal Article
• Authors: Zafred, Domen; Steiner, Barbara; Teufelberger, Andrea R.; Hromic, Altijana; Karplus, P. Andrew; Schofield, Christopher J.; Wallner, Silvia; Macheroux, Peter
• Is Part Of: FEBS journal. Volume 282:Number 16(2015)
• Page Start: 3060
• Page End: 3074
• Publication Date: 2015-02-24
• Subjects: Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.13212
• Languages: English
• ISSNs: 1742-464X

Abstract

<abstract abstract-type="main" id="febs13212-abs-0001"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="febs13212-sec-0001" sec-type="section"> <p>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.</p> </sec> <sec id="febs13212-sec-0002" sec-type="section"> <title>Database</title> <p>Structural data are available in PDB database under the accession numbers <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVE" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PVE</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVH" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PVH</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVJ" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PVJ</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVK" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PVK</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PWB" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PWB</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PWC" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PWC</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PZF" xlink:type="simple" xmlns:xlink="http://www.w3.org/1999/xlink">4PZF</ext-link> .</p> </sec> </abstract>