Summary for peptidase M09.001: bacterial collagenase V

Summary Alignment Tree Sequences Sequence features Distribution Literature Substrates Inhibitors

 

Names
MEROPS Namebacterial collagenase V
Other namesbacterial collagenase (Vibrio-type), collagenase Achromobacter iophagus, collagenase (Corynebacterium rathayii), collagenase (Empedobacter collagenolyticum), exopeptidase vcc (Vibrio cholerae), microbial collagenase (Vibrio type), vmc g.p. (Vibrio mimicus), VPM peptidase (Vibrio parahaemolyticus), vvpC g.p. (Vibrio parahaemolyticus 04)
Name and HistoryCollagenolytic activity in hide bacteria was first reported by Woods et al., 1972 and one of these bacteria was first identified as Achromobacter iophagus. Microbiological study later showed that this bacterium belongs to the largely marine genus Vibrio and accordingly it was renamed as Vibrio alginolyticus chemovar. Iophagus. This collagenolytic enzyme, quoted frequently under its historical name as Achromobacter collagenase or achromase, and more recently as Vibrio collagenase, is one of the most thoroughly studied bacterial collagenases.
Domain architecture
MEROPS Classification
Classification Clan MA >> Subclan MA(E) >> Family M9 >> Subfamily A >> M09.001
Holotypebacterial collagenase V (Vibrio alginolyticus), Uniprot accession P43154 (peptidase unit: 76-531), MERNUM MER0001172
History Identifier created: Handbook of Proteolytic Enzymes (1998) Academic Press, London.
Activity
Catalytic typeMetallo
PeplistIncluded in the Peplist with identifier PL00152
NC-IUBMBSubclass 3.4 (Peptidases) >> Sub-subclass 3.4.24 (Metalloendopeptidases) >> Peptidase 3.4.24.3
EnzymologyBRENDA database
PreparationThe enzyme protein is prepared from the culture medium and purified by DEAE-cellulose chromatography (Lecroisey et al., 1975) or followed by preparative gel electrophoresis (Tong et al., 1986). These reports indicate that there are several molecular forms of enzyme in the preparation; these are considered to be autodigestion products from a single polypeptide chain (Keil-Dlouha, 1976).
BiotechnologySeveral proposed uses.
SpecificityVibrio collagenase cleaves native collagen much more rapidly than does vertebrate interstitial collagenase (M10.001). In the fist step of degradation, this enzyme acts on collagen in a manner similar to the vertebrate collagenase, i.e. attacking at a point three-quarters of the way from the N-terminus, although the bond preferentially cleaved is different: Xaa+Gly instead of Gly+Leu or Gly+Ile (Lecroisey & Keil, 1979). Collagenase activity can be measured colorimetrically using a synthetic substrate (Pz-Pro-Leu+Gly-Pro-D-Arg from Sigma), and the assay system typically includes Tris–HCl buffer (pH 7.2; 50 mM), CaCl2 (1 mM) and NaCl (0.4 M). Vibrio and Clostridium collagenase cleave the same bond in the synthetic peptide Pz-Pro-Leu+Gly-Pro-D-Arg; however their specific activities are different: the purest Clostridium collagenase has a specific activity about 300 nkat mg−1 and Vibrio collagenase 1800 nkat mg−1 (Lecroisey et al., 1975, Keil-Dlouha, 1976). It is believed that a bacterial collagenase degrades exclusively collagen-like structures. Nevertheless, numerous proteins have short sequence stretches analogous to those in collagen. In fact, this collagenase has been successfully applied to produce highly specific cleavages in beta-casein (Gilles & Keil, 1976), myosin A, prolactin and adenylate kinase (Keil, 1992). The corresponding substrate subsites display a consistent characteristic pattern: Pro is either in position P2 or P2" and a small neutral residue (Gly, Ala) in P1 (Imhoff et al., 1992). Inhibitor analysis using synthetic peptides further reveals that the S3" subsite is important for substrate binding and the charged groups in the P3" position play a key role in the interaction of the inhibitors with enzyme, e.g. HS-CH2-CH2-CO-Pro-Arg was a much more potent inhibitor (Ki 0.5 μM) than HS-CH2-CH2-CO-Pro-Asp (Ki 70 μM) (Yiotakis & Dive, 1986).
pH optimumpH 6.0 (Keil et al., 1975)
Substrate commentsNative collagen
Inhibitor commentsThe enzyme activity is inhibited by EDTA, cysteine and histidine. and by the synthetic inhibitor HS-CH2-CH2-CO-Pro-Arg.
StructureVibrio collagenase is a protein of 81,875 Da and 739 amino acid residues as deduced from the DNA sequence analysis (Takeuchi et al., 1992). Collagenase protein is synthesized in precursor form (preprocollagenase), then the signal peptide and pro region are removed and the resulting mature form is secreted. The active enzyme contains 1 mol of zinc per mol enzyme (Keil-Dlouha, 1976) and there are two residues of His in the sequence -His-Glu-Tyr-Val-His- that may well be zinc ligands. The calculated pI from the amino acid composition is 4.4. Secondary structure comparison of this enzyme with thermolysin and Clostridium collagenase reveals that Vibrio collagenase contains a higher fraction of alpha helix than the Clostridium enzyme (Heindl et al., 1980).
LocationSecretable proteins from the bacterial cells.
PhysiologyRequired for bacterial nutrition.
Biological aspectsThis nonpathogenic bacterium was isolated from cured hides and shown to lyse collagen rapidly under aerobic conditions. The primary role of bacterial collagenase is the attack upon the host, destruction of the collagen barrier and provision of nutritional resources for bacteria. Vibrio collagenase is induced by the addition of collagen (Keil-Dlouha et al., 1976) or peptone (Reid et al., 1980) in the culture medium. There is a bacterial receptor for detecting collagen or peptides derived from collagen. This receptor is identified in membranes by a radioiodination reaction (Keil-Dlouha et al., 1983). Production of the enzyme is also regulated by temperature and oxygen (Hare et al., 1981). The use of Vibrio collagenase in tissue cell dispersions, elastin purification and selective cleavages of gene products in biotechnology is attractive. However, more important applications in human therapy are in the removal of necrotic tissues from burns, ulcers and decubitus ulcers because of its strong and specific activity against native collagen.
RNA splicingNo splicing and mRNA variants were known.
KnockoutThere are no report on the knockout experiment of the gene.
Pharmaceutical relevanceProposed as a potential agent for burn debridement.
Distinguishing featuresA quantitative difference exists between Vibrio and Clostridium collagenase in the action on a synthetic peptide, Pz-Pro-Leu+Gly-Ala-D-Arg, which is cleaved only by the Vibrio enzyme (Keil, 1992). Polyclonal antisera against the enzyme have been described (Fukushima et al., 1990, Choi et al., 1991), but are not commercially available.
Contributing authorsJun Fukushima, Department of Biotechnology, Akita Prefectural University, 241-438 Shimoshinjyou-Nakano, Akita 010-0195, Japan.