Production and characterization of two AA3 enzymes from Arabidopsis thaliana

Enzymes involved in the synthesis and degradation of complex carbohydrates are classified in the carbohydrate-active enzyme database (CAZy). One of the classes, auxiliary activities (AA), consists of redox enzymes that assist other carbohydrate-active enzymes. Within this class, the AA3 family, contains FAD-dependent oxidoreductases with diverse catalytic roles in fungi, where they participate in lignocellulose degradation. However, the biological functions of their plant counterparts remain largely unknown, and only a few plant AA3 enzymes have been characterized to date. As current industrial wood-processing methods do not fully utilize the potential of lignocellulosic biomass, there is growing interest in discovering new enzymes that could enhance lignocellulose conversion into valuable products. Plant AA3 oxidoreductases may represent an underexplored source of such biocatalysts, but a deeper understanding of their substrate specificities and catalytic properties is still required. This study presents the production and first characterization of two Arabidopsis thaliana AA3 enzymes, AtAA3C and AtAA3G. The enzymes were heterologously expressed in Pichia pastoris and purified using affinity chromatography. The activities of AtAA3C and AtAA3G were screened against a wide range of substrates using a high-throughput microplate assay. AtAA3C exhibited both oxidase and dehydrogenase activities toward aryl alcohols, with cinnamyl alcohol as its preferred substrate. It also displayed oxidase activity toward primary alcohols. AtAA3G exhibited oxidase activity, with the highest activity observed toward the long-chain alcohols 1-decanol and 1-dodecanol. The catalytic properties of these oxidases were further determined under optimal pH conditions using their preferred substrates. Compared to other characterized plant and fungal AA3 enzymes, AtAA3C and AtAA3G showed relatively low activities and catalytic velocities. This may reflect differences in their biological roles or that their true physiological substrates were not included in the tested substrate sets. Overall, these findings expand the current knowledge of plant AA3 enzymes and provide a basis for future studies.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2026

Author dc:creator

• Kiuru, Silja

• Arabidopsis thaliana
• AA3
• AtAA3C
• AtAA3G
• oxidoreductase
• enzyme characterization
• heterologous expression
• enzymes

Statement dc:rights

• CC BY-NC-ND 4.0

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/627069

OAI identifier oai:identifier

oai:helda.helsinki.fi:10138/627069