Cloning and sequencing of cDNA and genomic DNA encoding serine carboxypeptidase of Fusarium moniliforme that was copurified with phosphatase.
A previous study [Yoshida, H. et al., J. Biochem., 140, 813-823 (2006)] revealed that a protein of unknown nature was copurified with PDM phosphatase of Fusarium moniliforme. In this study, the identity of this protein was investigated. The results of homology search for the tryptic peptides derived from the purified preparation of PDM phosphatase strongly suggested that it might be serine carboxypeptidase. In fact, carboxypeptidase activity was demonstrated in the preparation and partial separation of carboxypeptidase from PDM phosphatase was achieved by gel filtration high-performance liquid chromatography. Cloning and sequencing of the full-length cDNA encoding the carboxypeptidase was successfully conducted. The cDNA possessed an open reading frame for a protein of 575 amino acid residues with a molecular mass of 64,650 Da, which was highly homologous to certain fungal serine carboxypeptidases. Comparison of the deduced amino acid sequence with the N-terminal sequence of the separated carboxypeptidase revealed that the mature enzyme starts at serine 56 of the precursor and has a molecular mass of 58,487 Da. Cloning and sequencing of the genomic DNA corresponding to the cDNA demonstrated that the gene of carboxypeptidase consists of four exons. A limited number of close homologs of F. moniliforme carboxypeptidase were detected among fungi by homology search and their evolutionary relationship was discussed.
Yoshida
Hiroshi
H
Department of Chemistry, Faculty of Medicine, Shimane University, 89-1 En-ya-cho, Izumo 693-8501, Japan. yoshidah@withe.ne.jp
Iizuka
Mari
M
Norioka
Naoko
N
Norioka
Shigemi
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D010744:Phosphoric Monoester Hydrolases; C019047:phosphodiesterase-phosphomonoesterase; D010727:Phosphoric Diester Hydrolases; D002268:Carboxypeptidases; C022633:serine carboxypeptidase