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

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. Database Structural data are available in PDB database under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVE, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVH, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVJ, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PVK, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PWB, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PWC and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4PZF .

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase

“…In the case of Pp BBE1, V175 is found in the pertinent ‘gate keeper’ position, and thus, the reduced enzyme is expected to rapidly react with dioxygen, as confirmed by the high rate of reoxidation, that is, 0.5 × 10 5 m ‐1 s ‐1 . In accordance with our ‘gate keeper’ model, replacement of this valine by leucine diminished the rate of reoxidation to 25 m −1 ·s −1 , thus basically suppressing the reaction of the reduced flavin with dioxygen as previously found for other members of this enzyme family .…”

Structure and biochemical properties of recombinant human dimethylglycine dehydrogenase and comparison to the disease‐related H109R variant

“…This gatekeeper appears to control access to an oxyanion hole essential to stabilize reaction intermediates during oxidation of the reduced isoalloxazine ring by dioxygen. In this family, valine was found to grant access to the oxyanion hole, whereas isoleucine denies access . Similarly, Leferink et al .…”

“…The direct comparison of a dehydrogenase (hDMGDH) and an oxidase ( Ag DMGO) allowed new insights into the structural elements involved in oxygen reactivity and supports concepts that are based on gatekeeper residues in the vicinity of the isoalloxazine ring system. In this vein, we show that a previously proposed model to rationalize oxygen reactivity in the vanillyl oxidase family is applicable to the family of sarcosine and dimethylglycine dehydrogenases and oxidases, respectively .…”

“…This gatekeeper appears to control access to an oxyanion hole essential to stabilize reaction intermediates during oxidation of the reduced isoalloxazine ring by dioxygen. In this family, valine was found to grant access to the oxyanion hole, whereas isoleucine denies access [25]. Similarly, Leferink et al discovered that a large number of oxidases in the VAO family contain either a glycine or a proline at a structurally conserved position near the isoalloxa- zine ring [26].…”

Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism

“…They identified a gatekeeper residue in the barberine bridge enzyme (BBE) and BBE-like enzyme Phl p4. When targeted in Phl p4 (I153V) oxidase activity was increased 60,000 fold; an inverse exchange in the related position of BBE (V169I) reduced oxidase activity by ca 500-fold [29]. Through biochemical and structural analysis the authors concluded that these enzymes have a cavity close to the flavin ring that accommodates dioxygen.…”