Caffeylpyruvate hydrolase from the bioluminescent fungus Neonothopanus gardneri is the key recycling enzyme in the fungal bioluminescence pathway

Recent studies uncovered the fungal bioluminescence pathway (FBP), a biosynthetic signaling axis to which caffeic acid is central. A key step, the hydrolysis of oxyluciferin into caffeic and pyruvic acids, was previously observed only in crude extracts, with pyruvate formation uncertain. Using gene homology, we identified and cloned cph from Neonothopanus gardneri, encoding a predicted fumarylacetoacetate hydrolase (FAH)-family enzyme, named ngarCPH. We successfully expressed and purified ngarCPH and demonstrated its catalytic activity on oxyluciferin, confirming formation of both products. Kinetic characterization revealed the optimal temperature and pH, while structural modeling and multiple sequence alignment indicate characteristics similar to those of FAH enzymes. This work not only expands the knowledge of the function of enzymes from the FAH superfamily but also confirms a complete, self-sustained bioluminescence cycle in fungi. Our findings support the future development of autonomous bioluminescent systems in heterologous hosts.