Identification of patulin-degrading enzyme in Geotrichum candidum XG1 based on proteomics and molecular dynamics...

Patulin (PAT) is a prevalent mycotoxin and environmental contaminant that poses serious threats to food safety, ecosystem stability, and human health. While previous studies have shown that intracellular enzymes from Geotrichum candidum XG1 can detoxify PAT, the specific enzyme responsible remained unidentified. In this study, proteomic analysis of Geotrichum candidum XG1 under PAT exposure revealed significant upregulation of redox-related proteins, implicating them in PAT detoxification. Molecular docking identified Geotrichum candidum XG1 aldo-keto reductase (GCAKR,initially annotated as YJR096W) as the top candidate enzyme, exhibiting the highest predicted binding affinity for PAT. Molecular dynamics simulations demonstrated the stability of the GCAKR-PAT complex, with a total binding energy of -16.57 kcal/mol. Key molecular components contributing to this stability included the ligand B:MOL-301, as well as protein residues TYR209 and PHE25. GCAKR was subsequently heterologously expressed in Escherichia coli, purified, and biochemically characterized. GCAKR exhibited optimal degradation activity at pH 8.0 and 30 °C, required NADPH as a coenzyme (≥1.0 mM), and achieved 100% degradation of 10 μg/mL PAT within 12 h at a concentration of 100 μg/mL. Its degradation efficiency was affected by enzyme/substrate concentration, with substrate inhibition observed at PAT concentrations ≥ 20 μg/mL. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) analysis confirmed the conversion of PAT to E-ascladiol, a metabolite with low toxicity and non-carcinogenic properties. This study identifies GCAKR as the primary PAT-degrading enzyme in Geotrichum candidum XG1 and offers a mechanistic foundation for environmentally friendly mycotoxin detoxification strategies in biological systems.