Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu + import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1 , strongly induced during Cu limitation and which encodes a protein related to Lytic Polysaccharide Monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient and Bim1 function in Cu accumulation depends upon Cu 2+ coordination and cell surface association via a GPI anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a new role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

The ability of the human fungal pathogen Cryptococcus neoformans to adapt to variable copper (Cu) environments within the host is key for successful dissemination and colonization. During pulmonary infection, host alveolar macrophages compartmentalize Cu into the phagosome and C. neoformans Cu-detoxifying metallothioneins, MT1 and MT2, are required for survival of the pathogen. In contrast, during brain colonization the C. neoformans Cu importers Ctr1 and Ctr4 are required for virulence. Central for the regulation and expression of both the Cu detoxifying MT1/2 and the Cu acquisition Ctr1/4 proteins is the Cu-metalloregulatory transcription factor Cuf1, an established C. neoformans virulence factor. Due to the importance of the distinct C. neoformans Cu homeostasis mechanisms during host colonization and virulence, and to the central role of Cuf1 in regulating Cu homeostasis, we performed a combination of RNA-Seq and ChIP-Seq experiments to identify differentially transcribed genes between conditions of high and low Cu. We demonstrate that the transcriptional regulation exerted by Cuf1 is intrinsically complex and that Cuf1 also functions as a transcriptional repressor. The Cu- and Cuf1-dependent regulon in C. neoformans reveals new adaptive mechanisms for Cu homeostasis in this pathogenic fungus and identifies potential new pathogen-specific targets for therapeutic intervention in fungal infections.

Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum (Mo) in Moco is the pyranopterin dithiolene (molybdopterin, MPT); however, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.