Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors

Abstract

Melanins are a class of natural pigments associated with a wide range of biological functions, including microbial virulence, energy transduction, and protection against solar radiation. Because of their insolubility and structural heterogeneity, solid-state nuclear magnetic resonance (NMR) spectroscopy provides an unprecedented means to define the molecular architecture of these enigmatic pigments. The requirement of obligatory catecholamines for melanization of the pathogenic fungus Cryptococcus neoformans also offers unique opportunities for investigating melanin development. In the current study, pigments produced with L-dopa, methyl-L-dopa, epinephrine, and norepinephrine precursors are compared structurally using (13)C and (1)H magic-angle spinning (MAS) NMR. Striking structural differences were observed for both aromatic and aliphatic molecular constituents of the mature fungal pigment assemblies, thus making it possible to redefine the molecular prerequisites for formation of the aromatic domains of insoluble indole-based biopolymers, to rationalize their distinctive physical characteristics, and to delineate the role of cellular constituents in assembly of the melanized macromolecules with polysaccharides and fatty acyl chain-containing moieties. By achieving an augmented understanding of the mechanisms of C. neoformans melanin biosynthesis and cellular assembly, such studies can guide future drug discovery efforts related to melanin-associated virulence, resistance to tumor therapy, and production of melanin mimetics under cell-free conditions.

Figure 1

150 MHz CPMAS solid-state ¹³C spectra of catecholamine precursors and the resulting C. neoformans melanins obtained by in vitro biosynthesis. Chemical shift differences among phenol carbons in the precursors (~140-150 ppm) are attributed to variations in hydrogen-bonding networks and crystal packing in the solid state.

Figure 2

Solid-state CPMAS ¹³C spectra of L-dopa C. neoformans melanins produced, as noted, with different isotopically enriched glucose sources and cell lines. The ¹³C spectra were acquired at 188 MHz (top and middle) and at 225 MHz (bottom). The resonances at 128 ppm are normalized to one another.

Figure 3

Solid-state MAS ¹H spectra of C. neoformans (CN) melanins produced from natural abundance L-dopa, methyl-L-dopa, norepinephrine and epinephrine, acquired at a ¹H frequency of 600 MHz. A spinning rate of 35 kHz was used to acquire spectra for all materials except the norepinephrine melanin, which was examined at 10 kHz.

Figure4

Proposed intermediates for biosynthesis of C. neoformans melanins from obligatory catecholamine substrates.