The exocellular beta-lactamase of Streptomyces albus G.Purification, properties and comparison with the exocellular DD-carboxypeptidase

in Biochemical Journal (1981), 193(1), 75-82

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric ... [more ▼]

The exocellular beta-lactamase of Streptomyces albus G has been purified to near protein homogeneity. It consists of one single polypeptide chain of mol.wt. 30 000-31 000, has a rather low isoelectric point (at pH 6.0) and contains less lysine (2.1%) and more half-cystine residues than most beta-lactamases from other Gram-positive bacteria. Penicillins are much better substrates than delta 3-cephalosporins; the catalytic-centre activity of good penicillin substrates is 333-500 s-1. The exocellular, mol.wt. 17 000 DD-carboxypeptidase of S. albus G [previously purified to protein homogeneity; Duez, Frere, Geurts, Ghuysen, Dierickx & Delcambe (1978) Biochem. J. 175, 793-800] behaves as an exceedingly poor beta-lactamase, hydrolysing benzylpenicillin into benzylpenicilloate 5 x 10(-6)-fold less rapidly than does the exocellular beta-lactamase. To all appearances, the beta-lactamase has no bivalent cation requirement whereas, as shown elsewhere [Dideberg, Charlier, Dupont, Vermeire, Frere & Ghuysen (1980) FEBS Lett. 117, 212-214, and Dideberg, Joris, Frere, Ghuysen, Weber, Robaye, Delbrouck & Roelands (1980) FEBS Lett. 117, 215-218], the DD-carboxypeptidase possesses one essential Zn2+ ion per molecule. Peptide 'mapping' and immunological studies suggest that the two Streptomyces enzymes probably have very different structural and mechanistic properties. [less ▲]

The penicillin-binding proteins in Streptococcus faecalis ATCC 9790

in European Journal of Biochemistry (1980), 110(2), 445-456

Streptococcus faecalis ATCC 9790 possesses seven membrane-bound penicillin-binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity ... [more ▼]

Streptococcus faecalis ATCC 9790 possesses seven membrane-bound penicillin-binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity profiles for 15 different beta-lactam antibiotics and stability under various conditions. In water and at 37 degrees C, all the native penicillin-binding proteins have half-lives longer than 20 h except protein 3b (half-life of about 600 min) and protein 4 (half-life of about 175 min). The short-lived 80 000-Mr protein 4 is spontaneously converted into a 73 000-Mr water-soluble, penicillin-binding protein 4. Similarly, the short-lived 82 000-Mr protein 3b seems to be the protein from which the 72 000-Mr water-soluble protein X spontaneously originates during incubation of the membranes. Release of both proteins 4 and X from the membrane is maximal under alkaline conditions; it is not inhibited by various protease inhibitors. After exposure to trypsin, the 43 000-Mr membrane-bound penicillin binding protein 6 (a DD-carboxypeptidase) gives to a 30 000-Mr water-soluble protein 6. Like the parent protein, protein 6 exhibits both DD-carboxypeptidase activity and penicillin-binding ability. With proteins 6 and 6, low dose levels of p-chloromercuribenzoate prevent both enzyme activity and combination with penicillin, thus strongly suggesting that a thiol group is involved in the enzyme active center. We have shown previously [Coyette et al. in Eur. J. Biochem. 88, 297--305 (1978) and 75, 231--239 (1977)] that the DD-carboxypeptidase protein 6 fragments the benzylpenicillin molecule with formation of phenylacetylglycine. Breakdown of the complex formed between [14C]benzylpenicillin and 14 000-Mr membrane-bound protein 1 is also 'enzyme-catalysed'. Most likely, however, the released product is penicilloate. With all the other penicillin-binding proteins whose molecular weights are intermediate between those of proteins 1 and 6, breakdown of the complexes formed with [14C]benzylpenicillin results from proteolysis and is not due to the release of the bound metabolite. None of the penicillin-binding proteins behaves, by itself, as a lethal target for beta-lactam antibiotic action on the living cells. [less ▲]

The active centres in penicillin-sensitive enzymes

in Philosophical Transactions : Biological Sciences (1980), 289(1036), 285-301

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site ... [more ▼]

The interaction between beta-lactam antibiotics and the penicillin-sensitive enzymes is a multiple-step process. Binding of the beta-lactam ring of the penam (or 3-cepham) nucleus occurs at binding site no. 1. Interaction between the N-14 substituent of the bound molecule and binding site no. 2 induces changes in binding site no. 1. In turn, the catalytic site thus created increases the chemical reactivity of the beta-lactam amide bond. As the beta-lactam ring opens and acylates an enzyme serine residue, the interaction between the thiazolidine (or dihydrothiazine) ring and binding site no. 3 stabilizes the acyl-enzyme complex. Enzyme regeneration slowly proceeds either by direct elimination of the penicilloyl moiety or via C-5-C-6 splitting of the bound metabolite. The fragment arising from thiazolidine yields free N-formyl-D-penicillamine while the enzyme-linked N-acylglycyl fragment is immediately attacked by an exogenous nucleophile correctly positioned on the acceptor site. Similarly, the enzyme action on L-X-D-Ala-D-Ala terminated peptides is mediated via a binding site no. 1 that combines with D-Ala-D-Ala, a binding site no. 2 that interacts with the side chain of the preceding L-residue, an inducible catalytic site and an acceptor site. Enzymes are known that form a transitory L-X-D-Ala-enzyme complex where the acyl group is ester-linked to the same serine residue as that involved in the formation of the penicilloyl-enzyme complex (Waxman et al., this symposium). Other enzymes, however, may function as catalyst templates. Depending on the enzymes, the independence of the beta-lactam and L-X-D-Ala-D-Ala active centres is more or less pronounced. [less ▲]

The 4.5 A resolution structure analysis of the exocellular DD-carboxypeptidase of Streptomyces albus G.

in FEBS Letters (1980), 117(1), 212-214

Effects of nucleophiles on the breakdown of the benzylpenicilloyl-enzyme complex EI formed between benzylpenicillin and the exocellular DD-carboxypeptidase--transpeptiase of Streptomyces strain R61

in Biochemical Journal (1979), 177(3), 909-916

Serine is one of the enzyme residues with which benzylpenicillin collides as a result of its binding to the Streptomyces strain-R61 DD-carboxypeptidase-transpeptidase enzyme. Nucleophilic attack occurs on ... [more ▼]

Serine is one of the enzyme residues with which benzylpenicillin collides as a result of its binding to the Streptomyces strain-R61 DD-carboxypeptidase-transpeptidase enzyme. Nucleophilic attack occurs on C(7) of the bound antibiotic molecule with formation of a benzylpenicilloyl-serine ester linkage, i.e. formation of the benzylpenicilloyl-enzyme EI complex. To reject the bound penicilloyl moiety and consequently to recover its initial activities, the strain-R61 enzyme has developed two possible mechanisms. Pathway A is a direct attack of the serine ester linkage by an exogenous nucleophile, resulting in the transfer of the benzylpenicilloyl moiety to this nucleophile. In pathway B, the benzylpenicilloyl moiety is first fragmented by C(5)-C(6) cleavage and the enzyme-bound phenylacetylglycyl residue thus produced is in turn transferred to the nucleophile. Pathway B occurs with water, glycylglycine and other amino compounds. Both pathways A and B occur with glycerol, other ROH nucleophiles and neutral hydroxylamine. The nucleophilic attacks are enzyme-catalysed. [less ▲]

Crystallographic data for a penicillin receptor : exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61

in Journal of Molecular Biology (1979), 127(2), 217-218

A pencillin-sensitive enzyme, the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7•6. X-ray ... [more ▼]

A pencillin-sensitive enzyme, the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7•6. X-ray examination of the orthorhombic crystals shows the space group is P212121, with unit cell dimensions a = 51•1 Å, B = 67•4 Å, and C = 102•9 Å. With four molecules of molecular weight 38,000, the Å3/dalton ratio for the cell is 2•33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2•4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å. [less ▲]

Interaction between penicillin and its enzyme target

in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (1979), 2

Stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid in relation to its possible occurrence as a degradation product of penicillin by the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61 and the membrane-bound dd-carboxypeptidase from Bacillus stearothermophilus

in Journal of Biological Chemistry (1978), 253(10), 3660-3665

The stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid has been studied under various conditions. In 10 mM cacodylate, pH 6.5, and at 55 degrees C, D-5,5-dimethyl-delta2-thiazoline-4 ... [more ▼]

The stability of D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid has been studied under various conditions. In 10 mM cacodylate, pH 6.5, and at 55 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) is hydrolyzed into N-formyl-D-penicillamine with a half-life of 3 to 4 min. On this basis, it is very unlikely that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid could be one of the end products resulting from the cleavage of benzylpenicillin by the DD-carboxypeptidase of Bacillus stearothermophilus (as reported by Hammarstrom and Strominger (1976) J. Biol. Chem. 251, 7947--7949). In 3 mM phosphate, pH 7.5, and at 37 degrees C, D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid (at concentrations lower than 1 mM) has a half-life of 45 min. On the basis of kinetic experiments carried out under these conditions with phenoxymethylpenicillin and the DD-carboxypeptidase-transpeptidase of Streptomyces R61, it is concluded that the primary product which arises from the thiazolidine moiety of the antibiotic molecule and gives rise to N-formyl-D-penicillamine, has a half-life of 10 min, a value which is not compatible with the hypothesis that D-5,5-dimethyl-delta2-thiazoline-4-carboxylic acid would be an intermediate involved in the fragmentation pathway. [less ▲]

Fragmentation of penicillin catalysed by the exocellular DD-carboxypeptidase-transpeptidase of Streptomyces strain r61. Isotopic study of hydrogen fixation on carbon 6

in FEBS Letters (1978), 88(1), 147-150

The exocellular DD-carboxypeptidase-endopeptidase from Streptomyces albus Gl. Purification and chemical properties

in Biochemical Journal (1978), 175(3), 793-800

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and ... [more ▼]

The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G was purified to protein homogeneity and compared with the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39. The S. albus G enzyme, as it is isolated, occurs in two forms. Enzyme I (30% of the total amount) and enzyme II (70% of the total amount) are identical in all respects, except that, by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, enzyme I has an apparent mol. wt. (9000) that is half of that found by molecular-sieve filtration under non-denaturing conditions. Irrespective of the technique used, enzyme II has an apparent mol. wt. of about 18500. [less ▲]