Fungal carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and play an essential role in fungal adaptation to environments with fluctuating CO2 concentrations. The opportunistic pathogen Candida parapsilosis expresses a single β-class CA, CpNce103p, which is structurally distinct from human α-class CAs and therefore may represent a promising antifungal target. Here, we report the biochemical and structural characterization of CpNce103p, including its crystal structure in complex with the classical CA inhibitor acetazolamide. Mass photometry and X-ray crystallography revealed that CpNce103p forms a stable homotetramer, similar to its homolog CaNce103p from C. albicans. Inhibition constants (Ki) were measured for a panel of 16 sulfonamide derivatives. One compound attained single-digit micromolar inhibition of CpNce103p, similar to the Ki of acetazolamide. Docking suggested the compound's binding mode, featuring zinc coordination and accommodation within the tight cavity. Our findings provide a structural basis for rational inhibitor design targeting fungal β-CAs and support CpNce103p as a viable target for the development of antifungal agents with selective activity.

Department of Biochemistry Faculty of Science Charles University Prague Hlavova 2030 128 43 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo náměstí 2 166 10 Prague Czech Republic

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