Peptide inhibitors of Streptomyces DD-carboxypeptidases

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms

Active-site-serine D-alanyl-D-alanine-cleaving-peptidase-catalysed acyl-transfer reactions. Procedures for studying the penicillin-binding proteins of bacterial plasma membranes

Under certain conditions, the values of the parameters that govern the interactions between the active-site-serine D-alanyl-D-alanine-cleaving peptidases and both carbonyl-donor substrates and beta-lactam suicide substrates can be determined on the basis of the amounts of (serine ester-linked) acyl-protein formed during the reactions. Expressing the 'affinity' of a beta-lactam compound for a DD-peptidase in terms of second-order rate constant of enzyme acylation and first-order rate constant of acyl-enzyme breakdown rests upon specific features of the interaction (at a given temperature) and permits study of structure-activity relationships, analysis of the mechanism of intrinsic resistance and use of a 'specificity index' to define the capacity of a beta-lactam compound of discriminating between various sensitive enzymes. From knowledge of the first-order rate constant of acyl-enzyme breakdown and the given time of incubation, the beta-lactam compound concentrations that are necessary to achieve given extents of DD-peptidase inactivation can be converted into the second-order rate constant of enzyme acylation. The principles thus developed can be applied to the study of the multiple penicillin-binding proteins that occur in the plasma membranes of bacteria

Secretion by Overexpression and Purification of the Water-Soluble Streptomyces K15 Dd-Transpeptidase/Penicillin-Binding Protein

Though synthesized with a cleavable signal peptide and devoid of membrane anchors, the 262-amino-acid-residue Streptomyces K15 DD-transpeptidase/penicillin-binding protein is membrane-bound. Overexpression in Streptomyces lividans resulted in the export of an appreciable amount of the synthesized protein (4 mg/litre of culture supernatant). The water-soluble enzyme was purified close to protein homogeneity with a yield of 75%. It requires the presence of 0.5 M-NaCl to remain soluble. It is indistinguishable from the detergent-extract wild-type enzyme with respect to molecular mass, thermostability, transpeptidase activity and penicillin-binding capacity

The peptidoglycan crosslinking enzyme system in Streptomyces R61, K15 and rimosus. Immunological studies

The exocellular DD-carboxypeptidases from Streptomyces R61, K 15, the lysozyme-releasable DD-carboxypeptidases from Streptomyces R61, K15 and rimosus, and the membrane-bound DD-carboxypeptidase of Streptomyces K15 are immunologically related to each other

Biochemistry of the Bacterial Wall Peptidoglycan in Relation to the Membrane

The peptidoglycan physically protects the membrane against deleterious influences. The membrane is involved in peptidoglycan synthesis and in other wall regulation mechanisms. The possible physiological function of a soluble carboxy-peptidase active on acyl-D-alanyl-D-R-OH substrates and its relation to the membrane-bound transpeptidase involved in peptidoglycan synthesis are discussed

Structure of the Cell Walls of Micrococcus lysodeikticus. III. Isolation of a New Peptide Dimer, Nα-[L-Alanyl-γ-(α-D-glutamyl-glycine )]-L-lysyl-D-alanyl-Nα- [L-alanyl-γ-(α-D-glutamyl-glycine)]-L-lysyl-D-alanine

The pentapeptide monomer Nα-[L-aIanyl-γ-(α-D-glutamyl-glycine)]-L-lysyl-D-alanine and two isomeric peptide dimers have been quantitatively isolated from walls of Micrococcus lysodeikticus. In one of the peptide dimers, referred as to peptide dimer (Ala→Lys), two pentapeptide monomers are linked through Nє-(D-alanyl)-L-lysine linkages. This linkage is hydrolyzed by the Streptomyces ML endopeptidase but not by the Myxobacter AL I protease. In the second peptide dimer, referred as to peptide dimer (Ala→Ala), two pentapeptide monomers are linked through D-alanyl-L-alanine linkages. This linkage is hydrolyzed by the Myxobacter AL I protease but not by the Streptomyces ML endopeptidase. According to the type of enzymatic degradation used, the pentapeptide monomer has been obtained in the free form, or substituted at its N-L-alanine terminus either by a D-lactic acid residue or by an N-acetylmuramic acid residue. Similarly, the peptide dimer (Ala→Ala) has been obtained in the free form or as a lactyl derivative. The peptide dimmer (Ala→Lys) has only been obtained in the free form. A comprehensive structure for a major part of the wall peptidoglycan is proposed. This structure takes into account the yields with which the peptide fragments are produced by the various enzymatic degradations. It provides explanation for the existence of a large number of peptide unsubstituted N-acetylmuramic acid residues in the glycan moiety. The structural peculiarity of the peptide moiety in M. lysodeikticus walls is the occurrence of large oligopeptides in which several pentapeptide monomers are linked through the aforementioned D-alanyl-L-alanine linkages. The isolation of the N-acetylmuramyl pentapeptide monomer involves, in one of the steps of the wall degradation, the use of a Streptomyces exo-N-acetylhexosaminidase active on both β-1,4-N-acetylglucosaminyl-N-acetylrnuramic acid and β-1,4-N-acetylmuramyl-N-acetylglucosamine disaccharides. This is the first known enzyme acting as an exo-β-N-acelylmuramidase

Streptomyces K15 DD-peptidase-catalysed reactions with suicide β-lactam carbonyl donors

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency