The role of the pro‐sequence in the processing and secretion of the thermolysin‐like neutral protease from <i>Bacillus cereus</i>

Wetmore, Diana; Wong, Sui‐Lam; Roche, Rodney S.

doi:10.1111/j.1365-2958.1992.tb00884.x

Cited by 59 publications

(38 citation statements)

References 31 publications

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“…Synthesis as an inactive precursor is a common feature of all extracellular proteases studied so far. The thermostable proteases from B. thennoproteolyticus (thermolysin) (32) and the thermolysin-related neutral proteases from B. stearothennophilus (31) and B. cereus (28,42) are synthesized as preproenzymes, as are the thermolabile neutral proteases A (44) and B (33) from B. subtilis and B.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Teufel

Götz

1993

J Bacteriol

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The gene sepA from Staphylococcus epidermidis TU3298-P, encoding the extracellular neutral metallopro- carnosus(pT181mcsPl) corresponded to the extracellular S. epidermidis T03298-P protease. The partially purified protease had a pH optimum between 5 and 7, and its activity could be inhibited by zinc-and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, indicating that it is a neutral metalloprotease. The protease had a low substrate specificity. Glucagon was cleaved preferentially between aromatic (Phe) and hydrophobic (Val) amino acids. The protease hydrolyzed casein and elastin. The amino acid sequence of the mature form of SepP1 revealed pronounced similarities with the thermolabile and thermostable neutral proteases of various bacilli (44 to 55% identity) and a central part of the mature form of the Pseudomonas aeruginosa elastase (31% identity). From homology comparison with the Baciflus thermoproteolyticus thermolysin, we predict that mature SepP1 binds one zinc ion at a conserved zinc-binding site.Most Staphylococcus aureus strains secrete proteolytic enzymes; three different types of proteases have been studied in detail. Serine protease from S. aureus V8, commonly referred to as V-8 protease or protease I, is an endoprotease which cleaves peptide bonds on the carboxy-terminal side of glutamic acid and to a lesser extent of aspartic acid (8). The enzyme, having a molecular weight of 12,000, is inhibited by diisopropyl fluorophosphate (DFP) but not by EDTA. Similar serine proteases with the same cleavage preference are also found in other S. aureus strains; however, they differ from the above-described enzyme in their size and sensitivity to DFP (2, 24). The amino acid sequence of a 27-kDa serine protease of S. aureus revealed a 43-residue C-terminal peptide composed nearly exclusively of aspartic acid, asparagine, and proline (6), proposed to be involved in the transport of the enzyme through the cytoplasmic membrane (7).The second type of protease from S. aureus V8, thiol protease (protease II), is characterized by an isoelectric point of pH 9.4, a molecular weight of 12,500, and a very broad specificity; it also exhibits esterase activity with N-benzoyl-L-tyrosine ethyl ester as a substrate (2). The enzyme is active only in the presence of reducing agents and is inactivated by heavy metals such as Hg2+, Zn2+, or Ag', indicating that protease II must possess free SH groups to be active and that it is a cysteine enzyme.The third protease of S. aureus V8, a metalloprotease (2), is completely inactivated by EDTA and is insensitive to sulfhydryl reagents and DFP. Calcium is essential for the stability of the protease. The molecular weight is 28,000, and the pH optimum for hydrolysis of casein is 7.4. Like the thiol * Corresponding author.protease, the metalloprotease has a rather broad proteolytic specificity, cleaving preferentially at the N-terminal side of hydrophobic residues (1). A metalloprotease with a molecular weight of 38,000 and a pH optimum of 7.0 has been isolated from a mut...

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Section: Discussionmentioning

confidence: 99%

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Teufel

Götz

1993

J Bacteriol

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“…3 and 4). The M4 enzymes are synthesized as pre-proproteins (5,6), with thermolysin itself having a 27-residue N-terminal pre-sequence, followed by a 204-residue prosequence and the 316-residue mature enzyme (7). As compared with other enzyme classes (reviewed in Refs.…”

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Intramolecular Processing of Prothermolysin

Marie-Claire

Roques

Beaumont

1998

Journal of Biological Chemistry

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Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M. J., and Beaumont, A. (1996) J. Biol. Chem. 271, 26477-26481). To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed in Escherichia coli. Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies. This was then solubilized in guanidinium hydrochloride, immobilized on a cobaltcontaining resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme. However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro. In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions. The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.Thermolysin (EC 3.4.24.27), a 34.6 kDa extracellular neutral protease produced by the Gram-negative Bacillus thermoproteolyticus, is the prototype member of the M4 family of zinc endopeptidases (1). This thermostable enzyme was the first zinc endopeptidase to be crystallized, and subsequent crystallographic studies have led to a mechanism of action being proposed for the zinc metallopeptidases in general (reviewed in Ref. 2). An active site model of thermolysin has also been used as a template for the design of inhibitors for several mammalian zinc metallopeptidases, for which structural data is lacking, such as neutral endopeptidase-24.11 (EC 3.4.24.11), angiotensin-converting enzyme (EC 3.4.15.1) and endothelinconverting enzyme (reviewed in Refs. 3 and 4). The M4 enzymes are synthesized as pre-proproteins (5, 6), with thermolysin itself having a 27-residue N-terminal pre-sequence, followed by a 204-residue prosequence and the 316-residue mature enzyme (7). As compared with other enzyme classes (reviewed in Refs. 8 and 9), there have been comparatively few studies on the roles of prosequences of bacterial zinc peptidases, although they have been shown to be essential for the production of active enzyme in vivo (10 -13) probably by facilitating folding (14). The prosequences, as independent polypeptides, can also act as inhibitors of their respective mature enzymes (14,15).From site-directed mutagenesis studies of the M4 enzymes, it has been inferred that prosequence processing is autocatalytic, as mutations of active site residues often result in low or negligible yields of mature enzyme (16 -19). In addition, certain mutations in the active site of thermolysin that change specificity lead to the production of more than one enzyme species differing by their N-terminals.1 For these reasons ...

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“…Class I propeptides generally function as intramolecular chaperones, facilitating folding of the mature protein. In some instances the propeptide can function in trans, but native conformation cannot be reached without the propeptide (21,36). Class I propeptides are primarily associated with proteases.…”

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The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

2005

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Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.

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The role of the pro‐sequence in the processing and secretion of the thermolysin‐like neutral protease from Bacillus cereus

Cited by 59 publications

References 31 publications

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis

Intramolecular Processing of Prothermolysin

The Metalloprotease ofListeria monocytogenesControls Cell Wall Translocation of the Broad-Range Phospholipase C

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