Purified Cryptococcus neoformans diphenol oxidase was further characterized and found to be a laccase based on substrate specificity. Dopamine was shown by mass spectroscopy to be oxidized by the purified enzyme to an intermediate of melanin synthesis. The enzyme contained four moles of copper and showed absorbance spectrum consistent with type I and II copper binding sites. Transcriptional activity of the gene was derepressed in the absence of glucose, conditions which also increased enzyme activity. The gene CNLAC1 was expressed in Pichia pastoris, producing diphenol oxidase activity as a secreted protein. A melanin negative strain of C. neoformans was complemented by transformation with a DNA construct containing a genomic fragment of CNLAC1. The complemented strain produced melanin pigment and had diphenol oxidase activity. CNLAC1 was disrupted using a genomic fragment containing CNLAC1 interrupted by the URA5 gene. The disrupted strain had no detectable diphenol oxidase activity and produced no pigment. Biochemical and molecular methods are being used to identify and isolate the first two enzymes in capsule polysaccharide synthesis in C. neoformans. The GDP-mannose pyrophosphorylase is being cloned by using homology found in the prokaryotic enzyme as a basis for PCR primer construction. The PCR product was subcloned, sequenced and used to identify a cDNA clone which is being sequenced. The second enzyme, x-1,3 mannosyltransferase in being purified using standard biochemical techniques. The enzyme has been identified using the photolabeling radioactive substrate 8-azido-GDP- mannose and localized in acrylamide gels. The isolated enzyme band will be analyzed for amino acid sequence. This sequence will be used to construct degenerate oligonucleotide probes with which to isolate a cDNA clone.
