Aug 2026· Biochemistry· Vol 65 16, pp.
2617-2628
· 0 citations· 60 references
Medicine
TL;DR
It is found that UmAA7s oxidize both chitooligosaccharides and their deacetylated forms (dCHOS) in an acetylation-site-dependent manner, extending the substrate scope of AA7s and laying the biochemical foundation for uncovering their biological functions during plant infection.
Abstract
Auxiliary activity family 7 (AA7) oxidoreductases are fungal flavoenzymes that catalyze the C1 oxidation of diverse oligosaccharides coupled to the reduction of molecular oxygen (oxidase activity) or organic molecules (dehydrogenase activity). These enzymes are predominantly derived from Ascomycota, with a smaller, less-explored fraction from Basidiomycota and Oomycota plant pathogens. Moreover, AA7 members are promising biocatalysts for the selective oxidation of carbohydrates, which is an enduring challenge in catalysis. However, the sequence space of AA7 enzymes remains largely uncharted, particularly within basidiomycota members, and the molecular determinants shaping substrate selectivity remain ill-defined. Focusing on the basidiomycete maize pathogen Ustilago maydis, we explored the boundaries of the AA7 sequence space by identifying and characterizing two enzymes, UmAA7A and UmAA7B, that are divergent from hitherto described members. We found that UmAA7s oxidize both chitooligosaccharides (CHOS) and their deacetylated forms (dCHOS) in an acetylation-site-dependent manner. The X-ray crystal structure of UmAA7A, sequence, and structural comparisons with a model of UmAA7B and other known CHOS-active AA7s, combined with docking analyses on CHOS and dCHOS, revealed specific active-site residues behind this unprecedented substrate specificity. Moreover, a previously not reported combination of a bicovalently tethered FAD and an atypical arrangement of residues at re-side of the FAD cofactor was associated with a mainly dehydrogenase activity profile, contrasting the majority of oxidases in AA7. Altogether, this work reveals the enzymatic oxidation of dCHOS, extending the substrate scope of AA7s and laying the biochemical foundation for uncovering their biological functions during plant infection.
Fungal phytopathogens represent a major threat to global agriculture, causing extensive yield losses. Among them, Colletotrichum spp. are infamous for their capacity of infecting a wide variety of monocot and dicot hosts. Motivated by the recent discovery in Colletotrichum orbiculare of a redox relay between Copper Radical Alcohol Oxidases from the AA5_2 subfamily and tandem peroxidases involved in plant infection, we investigated species from the Colletotrichum species complex (C. acutatum and C. tamarilloi) where the tandem peroxidase gene is absent. Instead, putative ring-cleavage dioxygenases are found within a conserved genomic locus adjacent to AA5_2 paralogues. Therefore, we hypothesized that ring-cleavage dioxygenases could function as redox partners for AA5_2. Given the limited information available on fungal dioxygenases, we performed a biochemical characterization of both Colletotrichum ring-cleavage dioxygenases. LC-MS analyses revealed that both enzymes catalyze intradiol (1,2-) ring cleavage of catecholic substrates and preferentially oxidize methyl-substituted catechols. Structural modeling indicated an open, solvent-exposed active site resembling that of spider mite intradiol ring cleavage dioxygenases and distinct from the compact, oligomeric bacterial homologs. However, both dioxygenases showed only a limited capacity to activate AA5_2 in vitro, indicating that close genomic proximity does not necessarily imply efficient functional coupling. Together, these biochemical insights into both AA5_2 and ring-cleavage dioxygenases provide a foundation for future studies aimed at elucidating their biological roles in fungal metabolism and pathogenicity.
Radka Končitíková, David Ribeaucourt, M. Haon et al.· The FEBS Journal· 0 citations
Sclerotinia sclerotiorum is a broad-spectrum soilborne fungal pathogen, causing substantial yield losses in crops worldwide. Auxiliary Activity Family 5 (AA5) copper radical oxidases (CROs) are members of the carbohydrate active enzymes (CAZymes), valued for their potential as biocatalysts. Although they were reported to contribute to fungal development and disease progression in some phytopathogenic fungi such as Colletotrichum graminicola, the roles of AA5 enzymes in S. sclerotiorum remain largely unexplored. Here, we systematically investigated S. sclerotiorum AA5 genes through reverse genetics and biochemical approaches. Phylogenetic analysis grouped the S. sclerotiorum AA5 proteins into two subfamilies, including three subfamily 1 (AA5_1) members and one subfamily 2 (AA5_2) member. Enzymatic characterization revealed that one AA5_1 enzyme, SsAA5c, exhibited the highest catalytic specificity towards D-glyceraldehyde compared with methylglyoxal and glycerol. In addition, a predicted peroxidase gene (SsPX1) was identified in tandem with the putative AA5_2 gene, SsAA5d. Phenotypic analysis of gene deletion mutants demonstrated that these genes differentially affect fungal development and virulence. While their exact physiological substrates await identification, our findings highlight important roles of AA5 oxidases and the associated peroxidase in the biology of S. sclerotiorum, providing insights that may contribute to future improved disease management strategies.
Jinyi Tan, Jessica K. Fong, H. Brumer et al.· Pathogens· 0 citations
Functional identification revealed that MDBE exhibits bifunctional hydrolytic activity, enabling it to hydrolyze both α-1,4 and α-1,6 glycosidic bonds, thereby allowing the degradation of both amylose and amylopectin.
Siting Feng, Peipei Peng, Jinsong Ouyang et al.· Applied Microbiology and Bio...· 0 citations
These findings define the core oxidative machinery underlying biomass deconstruction in T. reesei, revealing the major cellulose-oxidative role of TrLPMO9A and the importance of a cooperative redox network for efficient lignocellulose depolymerization.
Priscila T Rodrigues, C. R. Terrasan, N. Bulka et al.· Biotechnology for Biofuels a...· 0 citations
Mycolicibacterium
strains are among the most effective biofactories for converting phytosterols into active pharmaceutical intermediates. Despite the main route resembling fatty acid β-oxidation, the specific enzymes and their precise roles are poorly defined. In this study, we performed a comprehensive gene knock-out analysis in the industrial 4-AD-producing strain,
Mycolicibacterium neoaurum
HGMS9 strain, a variant of
M. neoaurum
B-3805. Our results demonstrate that the three β-oxidation cycles responsible for phytosterol side-chain degradation share a suite of core enzymes. Specifically, seven enzymes, including the acyl-CoA dehydrogenase ChsE1/E2, ChsE4/E5, the hydratase, ChsH1/H2 and the aldolase Ltp2, function in both the second and third β-oxidation cycles to generate the final products, 4-AD. Importantly, we identified five potential secondary routes that divert metabolism to produce eight off-route C22, C23 and C24 intermediates. These compounds represent valuable precursors for the synthesizing advanced steroids like corticosteroids and bile acids. This study not only clarifies the enzymatic steps of phytosterol side-chain degradation but also establishes a metabolic blueprint for engineering high-yielding
Mycolicibacterium
strains.
Vanadium-dependent haloperoxidases are enzymes found in bacteria, fungi and red or brown macroalgae. They are used as a defense system for the latter by producing bromoform. The intermediate product, HOBr or HOCl, is also a microbicidal compound that can be exploited in many domains as medical tools or disinfectant sprays for example. We show that the mutation identified in Ohshiro's work, which alters substrate specificity toward chloride, is transferable to the homologous enzyme from Chondrus crispus. We then applied genetic and enzymatic engineering to design a chimera with glucose oxidase, thereby enhancing antimicrobial properties by providing a local source of H2O2. We used the SpyTag/SpyCatcher technology to form the chimera and obtained homogenous objects, indicating that one oligomeric form is favored. We demonstrated the release of HOBr and HOCl thanks to NADH that reacts spontaneously with it outside the active site of the enzyme. We also measured the steady-state kinetic parameters of the wild-type or modified enzymes. The formation of the chimera increased the specificity of ccVHPO1 inside the chimera, towards KBr or H2O2, compared to the enzyme alone with the SpyCatcher. Finally, we showed a significant increase in the microbicidal effect between a coupled enzymatic system with the glucose oxidase (the two enzymes are free in solution) compared to the chimera system, which is completely bactericidal at concentrations around 20nM.
V. Amalric, S. Gounel, A. Thureau et al.· Journal of Biotechnology· 0 citations
