Diamondoid ketones as structural probes for active sites of fungal enzymes

• Cerrena zonata and Wolfiporia cocos showed the highest activity in submerged cultures and were selected for preparative-scale biotransformations. • Mild biocatalysis afforded valuable disubstituted adamantane and diamantane derivatives reported for the first time. • Cerrena zonata required substrate supplementation for oxidative activity. • Comparative transcriptomics of supplemented vs. non-supplemented Cerrena zonata cultures identified two candidate oxidative enzymes, supported by BLAST and molecular docking analyses. • The study enhances understanding of fungal oxidative pathways and supports future work toward enzyme characterization and heterologous expression in engineered hosts. To reveal the enzymes active sites, the oxidative activities of twelve wood-degrading fungi was tested using rigid adamantanone and diamantanone models that contain clearly defined hydrophobic and hydrophilic regions. Fungal cultures have been observed to exhibit three distinct catalytic behaviors, namely, exclusively carbonyl-reductive activity, exclusively C–H oxidative activity, or a combination of both. The two most promising fungi, Cerrena zonata and Wolfiporia cocos were selected for aerobic on-water preparative oxidations during which series of diols and hydroxyketones were produced and characterized. To identify the enzymes responsible for these transformations, experiments using fungal mycelia were performed. Mycelium of C. zonata exhibited oxidative activity only when the culture was supplemented with the model compound 24 h prior to mycelium separation; in the absence of supplementation, no activity was detected. In contrast, transformations using mycelium from both supplemented and non-supplemented cultures of W. cocos did not yield any products. To identify genes potentially involved in the biotransformation, a comparative transcriptomic analysis of supplemented and non-supplemented C. zonata cultures was performed. Based on BLAST analysis and molecular docking studies, two candidate enzymes potentially responsible for the oxidation process were identified. These findings lay the groundwork for unraveling the enzymatic mechanisms that drive hydrocarbon oxidation in wood-rot fungi and pave the way for future characterization and development of biocatalysts.