A unique signature identifies a family of zinc‐dependent metallopeptidases

Jongeneel, C. Victor; Bouvier, Jacques; Bairoch, Amos Marc

doi:10.1016/0014-5793(89)80471-5

Cited by 443 publications

(261 citation statements)

References 29 publications

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“…This histidine is within a zinc binding motif found in several metalloproteinases, but the zinc binding sequence of histatin 5 includes basic residues which are rare in non-conserved residue positions within and surrounding the consensus sequence [11]. Other histidyl C O1^H resonance changes also suggest site-speci¢c binding of zinc to histatin 5.…”

Section: Discussionmentioning

confidence: 98%

Zinc and copper bind to unique sites of histatin 5

et al. 2001

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Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side-chains of histatin 5 in aqueous solutions. Three C O1^H histidyl and the C Q^H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of C L^H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His-15 present within the^H^E^XX^H^z inc binding motif, and copper binding occurs within the N-terminal D^S^H^, ATCUN motif. ß

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Section: Discussionmentioning

confidence: 98%

Zinc and copper bind to unique sites of histatin 5

et al. 2001

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“…The region of highest identity is found at the N-terminal part of the enzyme. The encoded protein contains the signature sequence of the M1 family of zinc peptidases (HEXXH) (Jongeneel et al, 1989 ;Hooper, 1994) and this sequence was found in a region that is most conserved between ApsA, AAPI and LAPI (Fig. 1).…”

Section: Cloning Of the A Niger Apsa And Analysis Of The Genementioning

confidence: 99%

“…The majority of aminopeptidases belong to the M1 family of peptidases ; they are metalloenzymes (Van Wart, 1996) which require zinc for enzymic activity and share the zinc binding motif HEXXH (Jongeneel et al, 1989 ;Hooper, 1994).…”

Section: Introductionmentioning

confidence: 99%

Lysine aminopeptidase of Aspergillus niger The EMBL accession number for the sequence reported in this paper is AJ292570.

2001

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Conserved regions within the M1 family of metallo-aminopeptidases have been used to clone a zinc aminopeptidase from the industrially used fungus Aspergillus niger. The derived amino acid sequence of ApsA is highly similar to two yeast zinc aminopeptidases, LAPI and AAPI (53 3 and 50 9 % overall similarity, respectively), two members of the M1 family of metalloaminopeptidases. The encoding gene was successfully overexpressed in A. niger and the overexpressed product was purified and characterized. Aminopeptidase A was found to be active towards a number of amino acid p-nitroanilide (pNA) substrates, viz. K-pNA, R-pNA, L-pNA, M-pNA, A-pNA and F-pNA. The most preferred N-terminal amino acid is lysine and not leucine, arginine or alanine, the N-terminal amino acids preferred by the yeast homologues. The K m and K cat for K-pNA and L-pNA were 0 17 mM and 0 49 µkat mg N1 , and 0 16 mM and 0 31 µkat mg N1 , respectively. The pH optimum of the enzyme is between 7 5 and 8, whereas the enzyme is stable between pH 5 and 8. The enzyme is inhibited by the metal chelators EGTA, EDTA and 1,10-phenanthrolin. Bestatin was also able to inhibit the activity.

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“…In addition, there is homology among the majority of the eight proteins at five other positions in this region. Regions of this type have been proposed as a consensus region for recognizing members of the metalloproteinase family (McKerrow, 1987;Jongeneel et al, 1989). However, fibrolase and other venom-derived metalloproteinases do not appear to share significant additional sequence homology with other, well-characterized neutral metalloendopeptidases.…”

Section: A Randolph Et Afmentioning

confidence: 99%

Amino acid sequence of fibrolase, a direct‐acting fibrinolytic enzyme from agkistrodon contortrix contortrix venom

Randolph¹,

Chamberlain²,

Chu³

et al. 1992

Protein Science

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The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurusflavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of a-helix (4"70), but much greater 0-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine. Keywords: amino acid sequence; secondary structure; snake venom fibrolase Fibrinolytic activity has been found in the venom of snakes from the families Crotalidae, Viperidae, and Elapidae, with venom from members of the Crotalidae having the highest levels of fibrinolytic activity (for a recent review, see Markland, 1988). Fibrolase is a direct-acting fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom that does not require bloodborne cofactors for activity (Guan et al., 1991). As previously described (Markland, 1983;Markland et al., 1988; Reprint requests to: Anne Randolph, Chiron Research Laboratories, Retzios & Markland, 1988), the enzyme is a nonhemorrhagic, zinc metalloproteinase which cleaves primarily the a-chain of human fibrinogen and fibrin. Specific cleavage sites have been determined for several venom metalloproteinases using natural or synthetic substrates (Tu et al., 1981;Hagihara et al., 1985;Fox et al., 1986;Mori et al., 1987).

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A unique signature identifies a family of zinc‐dependent metallopeptidases

Cited by 443 publications

References 29 publications

Zinc and copper bind to unique sites of histatin 5

Zinc and copper bind to unique sites of histatin 5

Lysine aminopeptidase of Aspergillus niger The EMBL accession number for the sequence reported in this paper is AJ292570.

Amino acid sequence of fibrolase, a direct‐acting fibrinolytic enzyme from agkistrodon contortrix contortrix venom

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