title: Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase.
abstract: Melanin production is a major virulence factor for Cryptococcus neoformans, an organism causing life-threatening infections in an estimated 10% of AIDS patients. In order to characterize the events involved in melanin synthesis, an enzyme having diphenol oxidase activity was purified and its gene was cloned. The enzyme was purified as a glycosylated 75-kDa protein which migrated at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after deglycosylation by endoglycosidase F. Substrate specificity resembled that of a laccase in that it oxidized multiple diphenolic and diamino compounds. Dopamine was shown by mass spectroscopy to be oxidized to decarboxy dopachrome, an intermediate of melanin synthesis. The enzyme contained 4.1 +/- 0.1 mol of copper per mol. It resembled a laccase in its absorbance spectrum, containing a peak of 610 nm and the shoulder at 320 nm, corresponding to the absorbance of a type I and type III copper, respectively. The cloned gene of C. neoformans laccase (CNLAC1) contained a single open reading frame encoding a polypeptide 624 amino acids in length. The encoded polypeptide contained a presumptive leader sequence, on the basis of its relative hydrophobicity and by comparison of the sequence to that of the N-terminal sequence of the purified enzyme. CNLAC1 also contained 14 introns ranging from 52 to 340 bases long. Transcriptional activity of CNLAC1 was found to be derepressed in the absence of glucose and to correspond to an increase in enzymatic activity.
authors.affiliation: Clinical Mycology Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: P R
authors.initials: PR
authors.lastname: Williamson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D042845:Laccase; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D008667:Metalloproteins; D012333:RNA, Messenger; D010088:Oxidoreductases; D042845:Laccase