Methionine oxidation of Carbohydrate-Active enZymes during white-rot wood decay

Abstract

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well-documented, the impact of ROS on the oxidation of the secreted proteins remains unclear and the identity of the oxidized proteins is largely unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot model Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining labelled H218O2 with proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in other eukaryotes. We also evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay

“… Distribution of the identified secreted proteins and CAZymes. Values shown are the aggregate of all replicates for each day [ Table 1 ; Data Set 1 ( 34 )]. Bar graphs represent the number of CAZymes per class.…”

“…n.d., not detected. b The list of individual proteins and their abundance is provided in the Data Set 1 ( 34 ). …”

“…Values shown are the average of the three replicates ± standard deviation. ( B ) Met percentages of all proteins predicted to be secreted or intracellular in the P. cinnabarinus were compared with the Met percentages of experimentally validated intracellular and secreted proteins from fungi, metazoans, and plants [ Table 1 ; Data Set 1 ( 34 )]. Mean percentage of Met is indicated with black lines.…”

“…( A ) Total levels of Met oxidation as sulfoxide and sulfone, either 16 O or 18 O calculated using the spectral counts of all Met-containing peptides and compared to the level of non-oxidized Met for each time point [Table S2; Data Set 3 ( 34 )]. (B ) Total levels of Pro oxidation as glutamic 5-semialdehyde (G5S) and pyroglutamic acid (PA), either 16 O or 18 O calculated using the spectral counts of all Pro-containing peptides and compared to the level of non-oxidized Pro for each day [Table S3; Data Set 4 ( 34 )]. (C ) %NSAF of the catalase A0A060STN2 [Data Set 1 ( 34 )] and total H 2 O 2 -consuming activity at each time point.…”

“…(B ) Total levels of Pro oxidation as glutamic 5-semialdehyde (G5S) and pyroglutamic acid (PA), either 16 O or 18 O calculated using the spectral counts of all Pro-containing peptides and compared to the level of non-oxidized Pro for each day [Table S3; Data Set 4 ( 34 )]. (C ) %NSAF of the catalase A0A060STN2 [Data Set 1 ( 34 )] and total H 2 O 2 -consuming activity at each time point.…”