Bacterial Proteinases

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

In these p-helix structures the polypeptide chain is coiled into a wide helix, formed by p strands separated by loop regions. In the simplest form, the two-sheet p helix, each turn of the helix comprises two p strands and two loop regions (Figure 5.28). This structural unit is repeated three times in extracellular bacterial proteinases to form a right-handed coiled structure which comprises two adjacent three-stranded parallel p sheets with a hydrophobic core in between. 

To check if PemB is surface exposed, E. chrysanthemi cells were subjected to proteolysis. Treatment of the cell suspension with trypsin, proteinase K or chimotrypsin at a concentration of 0.1 to 1 mg/ml for 1 h did not cause PemB proteolysis or its liberation into the medium. Cell pre-treatment with EDTA-lysozyme, which renders the periplasmic proteins accessible to proteases, gave no effect. PemB was also resistant to proteolytic digestion in extract of cells disrupted by sonication or in a French press. Only addition of Triton X-100 (up to 0.1%) causing formation of the micelles with PemB lead to a quick proteolyis of this protein (data not shown). In another approach to analyse the PemB exposition, bacterial cells were labelled with sulfo-NHS-biotin. This compound is unable to cross membranes and biotinylation... 

C5a is inactivated by the myeloperoxidase-H202 system, which oxidises a methionine residue (Met 70) on the molecule group A streptococcal endo-proteinases also abolish chemotactic activity of C5a and related compounds. Neutrophil lysosomal enzymes (e.g. elastase and cathepsin G) also destroy C5a chemotactic activity, but as these proteases are inhibited by the serum antiproteinases, a -antiproteinase and a2-macroglobulin, the physiological role of neutrophilic proteases in the inactivation of C5a is questionable. Two chemotactic factor inactivators have been found in human serum an a-globulin that specifically and irreversibly inactivates C5-derived chemotactic factors, and a / -globulin that inactivates bacterial chemotactic factors. These activities are heat labile (destroyed by treatment at 56 °C for 30 min) and are distinct from those attributable to anaphylatoxin inactivator. An apparently specific inhibitor of C5-derived chemotactic activity has also been described in human synovial fluid and peritoneal fluid. This factor (molecular mass of 40 kDa) is heat stable and acts directly on C5a. 

Crystal structure of gingipain R an Arg-specific bacterial cysteine proteinase with a caspase-like fold. EMBOJ. 1999, 18, 5453-5462. 

Urokinase, t-PA, and streptokinase, a bacterial proteinase with similar activity, are used clinically to dissolve thrombi following heart attacks. All of these proteins are expressed recombinantly in bacteria (see p. 262). 

Driessen, F.M. (1989) Inactivation of lipases and proteinases (indigenous and bacterial), in Heat-induced Changes in Milk (ed. P.F. Fox), Bulletin 238, International Dairy Federation, Brussels, pp. 71-93. 

The gross proteolysis of casein is probably due solely to rennet and plasmin activity (O Keeffe et al. 1978). Bacterial proteases and peptides are responsible for subsequent breakdown of the large peptides produced by rennet and plasmin into successively smaller peptides and finally amino acids (O Keeffe et al. 1978). If the relative rate of proteinase activity by rennet, plasmin, and bacterial proteases exceeds that of the bacterial peptidase system, bitterness in the cheese could result. Bitter peptides can be produced from a,-,- or /3-casein by the action of rennet or the activity of bacterial proteinase on /3-casein (Visser et al. 1983). The proteolytic breakdown of /3-casein and the subsequent development of bitterness are strongly retarded by the presence of salt (Fox and Walley 1971 Stadhouders et al. 1983). The principal source of bitter peptides in Gouda cheese is 3-casein, and more particularly the C-terminal region, i.e., 3(193-209) and 3(193-207) (Visser et al. 1983). In model systems, bitter peptides are completely debittered by a peptidases system of S. cremoris (Visser et al. 1983). 

Andrews, A. T. 1983B. Breakdown of caseins by proteinases in bovine milks with high somatic cell counts arising from mastitis or infusion with bacterial endotoxin. J. Dairy Res. 50, 57-66. 

Argyls, P. J., Mathison, G. E. and Chandan, R. C. 1976. Production of cell-bound proteinase by Lactobacillus bulgaricus and its location in the bacteried cell. J. Appl. Bacte-riol 41, 175-184. 

Several bacterial and tissue proteinases are able to generate the active di-chain neurotoxin (Dasgupta 1994 Krieglstein et al. 1991). The heavy chain (H, 100 kDa) and the light chain (L, 50 kDa) remain associated via noncovalent interactions and via the conserved interchain S-S bond, whose integrity is essential for neurotoxicity (Figure 1) (de Paiva et al. 1993 Schiavo et al. 1990 Simpson et al. 2004). 

M. Sasaki, H. Yamamoto, H. Yamamoto, and 5. lida. Interaction of human serum proteinase inhibitors with proteolytic enzymes of animal, plant, and bacterial origin. J. Biochem. 75 171 119741. 

Pancreatic enzymes, preferably trypsin, have been used for the chemical characterisation and identification of many known bioactive peptides. For example, ACE-inhibitory peptides as well as CPPs are most commonly produced by trypsin (Maruyama and Suzuki, 1982 Berrocal et al., 1989). On the other hand, other enzymes and different enzyme combinations of proteinases, including alcalase, chymotrypsin, pancreatin and pepsin, as well as enzymes from bacterial and fungal sources have also been utilised to generate bioactive peptides. Higher yields of CPPs and, particularly, higher amounts of asl-casein f(59-79) in the hydrolysate have been obtained with casein micelles successively digested with pepsin and trypsin... 

The iron complexes of both human serum transferrin and chicken ovo-transferrin were completely resistant to all proteolytic enzymes tested (7). The metal-free proteins, on the other hand, were rapidly hydrolyzed under similar conditions. Fig. 14 compares results with bovine a-chymo-trypsin. Other enzymes used with similar results were bovine trypsin, bacterial proteinase (Nagarse) and ficin (735). The weaker copper complex of chicken ovotransferrin was hydrolyzed, however, at a rate approx-... 

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