Biochemical characterisation of fungal bioluminescence enzymes reveals substrate inhibition and secondary turnover of 3-hydroxyhispidin by H3H

The fungal bioluminescence pathway enables autonomous light production from caffeic acid and has emerged as a versatile platform for bioimaging and synthetic biology. However, biochemical characterisation of its core enzymes remains limited. Here, we establish recombinant purification and quantitative in vitro characterisation of fungal luciferase (Luz) and hispidin-3-hydroxylase (H3H) homologues from Neonothopanus nambi and Mycena chlorophos. Complementary luminescence and mass spectrometry-based assays revealed pronounced substrate inhibition of H3H by hispidin, conserved across both homologues. Direct monitoring of substrate turnover further revealed an unexpected secondary H3H-catalysed conversion of 3-hydroxyhispidin that remained dependent on NADPH and FAD. NMR, isotope labelling and product characterisation support an additional oxidative transformation followed by formation of multiple downstream products, including caffeic acid, thereby reconnecting secondary turnover with an upstream intermediate of the pathway. H3H nevertheless retained a strong kinetic preference for hispidin over 3-hydroxyhispidin. Together, these findings reveal previously unrecognised catalytic complexity within the fungal bioluminescence pathway and provide a biochemical framework for understanding and engineering pathway flux.