title: Identification of a Caldariomyces fumago mutant secreting an inactive form of chloroperoxidase lacking the heme group and N-glycans.
abstract: By mutant colony screening of Caldariomyces fumago a mutant was isolated which was slightly greenish on fructose minimal medium and grew slower in comparison to the wild type. The supernatant samples lacked the Soret band typical for the heme group of the CPO and nearly no CPO activity was detected. SDS-PAGE analysis of mutant culture supernatant samples showed production of a 38-40 kDa protein while wild type samples contain the 42 kDa CPO protein. Protein identification using nanoLC-ESI-MS/MS was performed and based on three peptides the protein in the mutant culture was identified as CPO. No differences in the CPO gene sequences of wild type and mutant were found indicating a post-translational defect in protein maturation. Deglycosylation experiments using CPO from wild type and mutant were carried out. After removing N-linked oligosaccharides from wild type CPO a protein band at 38-40 kDa was detected. Our results reveal that the mutant protein lacks the heme group as well as the N-glycans.
authors.affiliation: DECHEMA Research Institute, Frankfurt am Main, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hüttmann
authors.forename: Markus
authors.initials: M
authors.lastname: Buchhaupt
authors.forename: Jens
authors.initials: J
authors.lastname: Schrader
mesh_terms: D001203:Ascomycota / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D002711:Chloride Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006418:Heme / Q000378:metabolism; D008970:Molecular Weight; D009154:Mutation; D036103:Nanotechnology / Q000379:methods; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D011134:Polysaccharides; D006418:Heme; D002711:Chloride Peroxidase