Development of difluoro-Kdn mechanism-based probes to label and visualize Kdnases in Aspergillus fumigatus

Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum, and several fungal species of the genus Aspergillus, including Aspergillus terreus and Aspergillus fumigatus.. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus (AfKdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed AfKdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.