Purification and characterization of Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene.
Clostridium perfringens type C NCIB 10662 produced various gelatinolytic enzymes with molecular masses ranging from approximately 120 to approximately 80 kDa. A 120-kDa gelatinolytic enzyme was present in the largest quantity in the culture supernatant, and this enzyme was purified to homogeneity on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was identified as the major collagenase of the organism, and it cleaved typical collagenase substrates such as azocoll, a synthetic substrate (4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg [Pz peptide]), and a type I collagen fibril. In addition, a gene (colA) encoding a 120-kDa collagenase was cloned in Escherichia coli. Nested deletions were used to define the coding region of colA, and this region was sequenced; from the nucleotide sequence, this gene encodes a protein of 1,104 amino acids (M(r), 125,966). Furthermore, from the N-terminal amino acid sequence of the purified enzyme which was found in this reading frame, the molecular mass of the mature enzyme was calculated to be 116,339 Da. Analysis of the primary structure of the gene product showed that the enzyme was produced with a stretch of 86 amino acids containing a putative signal sequence. Within this stretch was found PLGP, the amino acid sequence constituting the Pz peptide. This sequence may be implicated in self-processing of the collagenase. A consensus zinc-binding sequence (HEXXH) suggested for vertebrate Zn collagenases is present in this bacterial collagenase. Vibrio alginolyticus collagenase and Achromobacter lyticus protease I showed significant homology with the 120-kDa collagenase of C. perfringens, suggesting that these three enzymes are evolutionarily related.
Matsushita
O
O
Department of Microbiology, Kagawa Medical School, Japan.
Yoshihara
K
K
Katayama
S
S
Minami
J
J
Okabe
A
A
D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology / Q000235:genetics*; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000235:genetics* / Q000302:isolation & purification*; D016384:Consensus Sequence; D015342:DNA Probes; D005780:Gelatin / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism
D001391:Azo Compounds; D015342:DNA Probes; D010455:Peptides; C028470:Azocoll; D005780:Gelatin; D003094:Collagen; D017364:Collagenases; D015032:Zinc