Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae

Abstract

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k (cat)/k(m)), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.

The GMC superfamily of oxidoreductases revisited: analysis and evolution of fungal GMC oxidoreductases

“…Listed organisms are sorted according to their appearance in the tree (top to bottom). 1—Takakura and Kuwata [93]; 2—Artolozaga et al [3]; 3—Danneel et al [18]; 4—US Patent 6146865, 2000; 5—Maresova et al [61]; 6—Daniel et al [16]; 7—Vecerek et al [99]; 8—Bannwarth et al [6]; 9—Pisanelli et al [73]…”

The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

“…), while PDH expression is limited to the Agaricales [7], which are typical litter-degrading fungi that live in forest and grassland soil and are the primary decomposers of residual plant material (leaves, needles, twigs, bark, grass) in the uppermost soil layer. It should be noted that P2O-encoding genes were also found and experimentally confirmed in some members of the genus Aspergillus , which does not degrade lignocellulose, notably in species where a glucose 1-oxidase (GOX) is absent [8].…”

Repurposing Inflatable Packaging Pillows as Bioreactors: a Convenient Synthesis of Glucosone by Whole-Cell Catalysis Under Oxygen

“…Pyranose 2-oxidases are found in diverse basidiomycetous fungi [19,24], and a subset of these fungi also express pyranosone dehydratase, which converts glucosone to cortalcerone that is proposed to function as an antibacterial agent suppressing competition for wood decay fungi [25,26]. In addition to decay wood basidiomycetes, POX has also been characterized from ascomycetes [27] and bacteria [28,29], and therefore, there is a broader context of possible catalysts that might be used for sugar oxidations with molecular oxygen. For the present study, we use E. coli whole-cell catalysis with recombinant POX from the white-rot wood decay fungus Phanerochaete chrysosporium.…”