Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H ₂ O ₂ in Brown Rot Decay of Wood

Abstract

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K m ‫؍‬ 2.3 mM, k cat ‫؍‬ 15.6 s ؊1 ). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H 2 O 2 , a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

“…Similarly, methanol released via demethoxylation of lignin (3,4) may play an important role in H 2 O 2 generation as a substrate for methanol oxidase. Such a role is consistent with our observed expression patterns and with previous investigations with G. trabeum (11). Of course hydroxyl radical may also play an important role early in decay, and it has been demonstrated to preferentially attack hemicellulose in wood (30).…”

Furfurylated wood: impact onPostia placentagene expression and oxalate crystal formation

“…1). Previous immunolocalization studies implicated the G. trabeum alcohol oxidase as a potential source of H 2 O 2 (Daniel et al 2007) It has been proposed that phenol oxidase enzymes, in addition to reducing toxic quinones in the fungal environment, are involved in the reduction of quinones in the chelator-mediated Fenton system (Goodell et al 1997a;Arantes et al 2012) as the oxidised chelators diffuse back to the cell lumen, and thereby perpetuate Fenton reactions.…”

Bioethanol: Feedstock Alternatives, Pretreatments, Lignin Chemistry, and the Potential for Green Value-Added Lignin Co-Products

“…Oxalate decarboxylase is common expressed in WRF to maintain stable concentrations of oxalic acid, which, at high concentrations, inhibits lignolytic peroxidases. Other enzymes including alcohol oxidase isolated from G. trabeum and P. placenta are also capable of producing hydrogen peroxide [144,164]. Demethylation of lignin can produce methanol, which is toxic to fungi.…”

“…Demethylation of lignin can produce methanol, which is toxic to fungi. Both WRF and BRF are capable of demethylating lignin and appear to efficiently express alcohol oxidases [19], which have a high affinity for methanol [164]. In the presence of alcohol oxidases, methanol is converted to formaldehyde generating H 2 O 2 in the process [164].…”

“…Both WRF and BRF are capable of demethylating lignin and appear to efficiently express alcohol oxidases [19], which have a high affinity for methanol [164]. In the presence of alcohol oxidases, methanol is converted to formaldehyde generating H 2 O 2 in the process [164]. Glyoxal oxidase (GLOX) is another common H 2 O 2 producing enzyme which functions by catalyzing the oxidation of aldehydes to carboxylic acids concomitantly reducing molecular oxygen to hydrogen peroxide by the following reaction [165] These represent only a few of the mechanisms that have been characterized through which fungi produce H 2 O 2 .…”