Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC.
Two new proteinases secreted by Cryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that the eapB and eapC genes contain three and two introns, respectively. The products of the eapB and eapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. The eapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of the eapC coding sequence in an endothiapepsin-deficient (EapA-) C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.
Jara
P
P
Sanofi Recherche, Centre de Labège, France.
Gilbert
S
S
Delmas
P
P
Guillemot
J C
JC
Kaghad
M
M
Ferrara
P
P
Loison
G
G
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000235:genetics*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification; D004792:Enzyme Precursors / Q000235:genetics*; D005656:Fungal Proteins; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid
D004271:DNA, Fungal; D004792:Enzyme Precursors; D005656:Fungal Proteins; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C097995:EapB protein, Cryphonectria parasitica; C097996:EapC protein, Cryphonectria parasitica