Molecular cloning and biochemical characterization of a Cu,Zn-superoxide dismutase from Scedosporium apiospermum.
A Cu,Zn-superoxide dismutase has been characterized from Scedosporium apiospermum, a fungus which often colonizes the respiratory tract of patients with cystic fibrosis. Enzyme production was stimulated by iron starvation. Purification was achieved from mycelial extract from 7-day-old cultures on Amberlite XAD-16. The purified enzyme presented a relative molecular mass of 16.4 kDa under reducing conditions and was inhibited by potassium cyanide and diethyldithiocarbamate, which are two known inhibitors of Cu,Zn-SODs. Its optimum pH was 7.0 and the enzyme retained full activity after pretreatment at temperatures up to 50 degrees C. Moreover, a 450-bp fragment of the gene encoding the enzyme was amplified by PCR using degenerate primers designed from sequence alignment of four fungal Cu,Zn-SODs. Sequence data from this fragment allowed us to design primers which were used to amplify by walking-PCR the flanking regions of the known fragment. SaSODC gene (890 bp) corresponded to a 154 amino acid polypeptide with a predicted molecular mass of 15.9 kDa. A database search for sequence homology revealed for the deduced amino acid sequence 72 and 83% identity rate with Cu,Zn-SODs from Aspergillus fumigatus and Neurospora crassa, respectively. To our knowledge, this enzyme is the first putative virulence factor of S. apiospermum to be characterized.
Lima
Osana C
OC
Groupe d'Etude des Interactions Hôte-Parasite, UPRES-EA 3142, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, 4, rue Larrey, 49933 Angers Cedex 9, France.
Larcher
Gérald
G
Vandeputte
Patrick
P
Lebouil
Anne
A
Chabasse
Dominique
D
Simoneau
Philippe
P
Bouchara
Jean-Philippe
JP
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D021681:Scedosporium / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc