Cryptococcus neoformans laccase catalyses melanin synthesis from both D- and L-DOPA

1 Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 2 Department of Biology, Farmingdale State College, 2350 Broadhollow Road, Farmingdale, NY 11735, USA 3 Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA Correspondence Arturo Casadevall casadeva{at}aecom.yu.edu The human fungal pathogen Cryptococcus neoformans produces melanin in the presence of various substrates, including the L enantiomer of 3,4-dihydroxyphenylalanine (DOPA). The enzyme laccase catalyses the formation of melanin by oxidizing L -DOPA, initiating a series of presumably spontaneous reactions that ultimately leads to the polymerization of the pigment in the yeast cell wall. There, melanin protects the cell from a multitude of environmental and host assaults. Thus, the ability of C. neoformans to produce pigments from a variety of available substrates is likely to confer a survival advantage. A number of C. neoformans isolates of different serotypes produced pigments from D -DOPA, the stereoisomer of L -DOPA. Acid-resistant particles were isolated from pigmented C. neoformans cells grown in the presence of D -DOPA. Biophysical characterization showed the particles had a stably detectable free-radical signal by EPR, and negative zeta potential, similar to L -DOPA-derived particles. No major differences were found between L - and D -DOPA ghosts in terms of binding to anti-melanin antibodies, or in overall architecture when imaged by electron microscopy. C. neoformans cells utilized L - and D -DOPA at a similar rate. Overall, our results indicate that C. neoformans shows little stereoselectivity for utilizing DOPA in melanin synthesis. The ability of C. neoformans to use both L and D enantiomers for melanization implies that this organism has access to a greater potential pool of substrates for melanin synthesis, and this could potentially be exploited in the design of therapeutic inhibitors of laccase. Abbreviations: DOPA, 3,4-dihydroxyphenylalanine