Jean-Marie Ghuysen

Enzymes that degrade bacterial cell walls

Publisher Summary This chapter focuses on the mechanism of enzymes that degrade bacterial cell walls. The cell walls of all bacteria are composed of two or more polymers. They are ubiquituous in the bacterial world, are responsible for the shape and strength of the walls, and allow the cells to live in environmental conditions which are hypotonic with respect to their high intracellular osmotic pressure. These polymers are a glycopeptide built up of polyacetylhexosamine and peptide chains, and the action of all bacteriolytic enzymes. The basic structure of the peptide subunits is probably similar to that found in uridine diphosphate (UDP)-acetylmuramyl-peptide cell wall precursors. Some or all of the peptide chains linked to adjacent polysaccharide backbones are in turn linked to each other, resulting in a network of at least two dimensions. The digestion of different bacterial walls by various bacteriolytic enzymes gives rise to fragments of highly diverse nature and molecular complexity. A lyric intrapeptide hydrolase induces the appearance of new terminal amino and carboxyl groups. The enzymes that degrade the glycopeptide of bacterial cell walls and of which the specificity is at least partially known, are tabulated in the chapter.

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan‐polymerizing penicillin‐binding protein 1b of Escherichia coli

The penicillin‐binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid‐transported N‐acetyl glucosaminyl‐β‐1,4‐N‐acetylmuramoyl‐l‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46‐N844) PBP1bγ. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46‐N844) PBP1bγ possesses an amino‐terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an ≅ 100‐amino‐acid insert, to a D198‐G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of ≅ 39 000 M−1 s−1. Glu‐233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu‐233 γ‐COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4‐OH group of a nucleophile N‐acetylglucosamine. Asp‐234 of motif 1 or Glu‐290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4‐OH group of the N‐acetylglucosamine. In turn, Tyr‐310 of motif 4 is an important component of the amino acid sequence‐folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447‐N844 acyl transferase module, which possesses the catalytic centre‐defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active‐site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser‐510‐assisted double‐proton shuttle are glycan strands substituted by cross‐linked tetrapeptide–pentapeptide and tetrapeptide–tetrapeptide dimers and uncross‐linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on d‐alanyl‐d‐alanine‐terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross‐linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

Comparison of the sequences of class A beta-lactamases and of the secondary structure elements of penicillin-recognizing proteins.

The sequences of class A beta-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The Staphylococcus aureus PC1 enzyme, although somewhat closer to the enzyme from the Bacillus group, did not belong to any of the groups of beta-lactamases. The similarities between the secondary structure elements of these enzymes and those of the class C beta-lactamases and of the Streptomyces sp. strain R61 DD-peptidase were also analyzed and tentatively extended to the class D beta-lactamases. A unified nomenclature of secondary structure elements is proposed for all the penicillin-recognizing enzymes.

A quasi-Newton algorithm for first-order saddle-point location

SummaryA new algorithm for the location of a transition-state structure on an energy hypersurface is proposed. The method is compared to three other quasi-Newton step calculations available in literature. Numerical results derived from several examples are compared to those obtained by the two algorithms implemented in the Gaussian package.

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506.

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin‐binding proteins (PBPs) which reduce their affinity for β‐lactam antibiotics. PBP2x is one of the high‐Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime‐resistant laboratory mutants. In this study, we have sequenced a 2564 base‐pair chromosomal fragment from the penicillin‐sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base‐pair open reading frame was found which coded for a protein having an Mr of 82.35 kD, a value which is in good agreement with the Mr of 80–85kD measured by SDS‐gel electrophoresis of the PBP2x protein itself. The N‐terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime‐resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the β‐lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from β‐lactam‐resistant strains are known. The penicillin‐binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N‐terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin‐resistant Staphylococcus aureus PBP2′. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low‐Mr PBPs.

Lack of Cell Wall Peptidoglycan versus Penicillin Sensitivity: New Insights into the Chlamydial Anomaly

Intracellular bacterial pathogens enter their hosts surrounded by a membrane-bound vacuole and use a panel of tricks to exploit or evade eukaryotic cell functions ([9][1],[12][2]). Chlamydia inhabits vesicles that do not fuse with lysosomes and remains within these parasitophorous vacuoles (termed

Acetylhexosamine compounds enzymically released from Micrococcus lysodeikticus cell walls: III. The structure of di- and tetra-saccharides released from cell walls by lysozyme and Streptomyces F1 enzyme

Two compounds, both of which have been isolated from cell walls of Micrococcus lysodeikticus digested with lysozyme or Streptomyces F1 enzyme, have been identified as di- and tetra-saccharides. The reducing groups of the di- and tetra-saccharides and those of the high-molecular weight, non-dialysable compounds belong to muramic acid. β-glucosidase yields free N-acetylglucosamine and N-acetylmuramic acid from the di- and tetra-saccharides. The proposed structure for the di-saccharide liberated by lysozyme and F1 is: 6-O-β-N-acetylglucosaminyl-N-acetylmuramic acid. The tetra-saccharides isolated from lysozyme and F1 digests appear to be identical. The structure proposed for the tetra-saccharide isolated from lysozyme digested walls is: O-β-N-acetylglucosaminyl-(I→6)-O-β-N-acetylmuraminyl-(I→4)-O-β-N-acetylglucosaminyl- (I→6)-β-N-acetylmuramic acid. Both lysozyme and Streptomyces F1 enzyme degrade di- and tetra-chitobiose, indicating their β(I→4) N-acetyl hexosaminidase activity.

Occurrence of a serine residue in the penicillin-binding site of the exocellular DD-carboxy-peptidase-transpeptidase from Streptomyces R61

According to the hypothesis proposed by Tipper and Strominger in 1965 for the mechanism of action of penicillin [ 11, the antibiotic, acting as a structural analogue of the substrate, would acylate an essential sulfhydryl group of an enzyme that catalyzes peptide crosslinking during wall peptidoglycan synthesis in bacteria. However, the experimental results presented in favour of the involvement of a sulfhydryl group in the transpeptidation reaction as well as in the binding of penicillin have never been very convincing. The exocellular DD-carboxypeptidase-transpeptidase that is excreted by Streptomyces R61 during growth, is very sensitive to penicillin [2] and appears to be a good model for the study of the interaction between the enzyme, substrates and antibiotic [3,4]. At 37°C the stoichiometric complex formed between benzylpenicillin and this enzyme decomposes with half-life of 80 min, yielding active enzyme, phenylacetylglycine and N-formyl-D-penicillamine [5,6] . More recently, however, it has been observed that when a solution of the [ 14C] benzylpeniciIlin-enzyme complex (in 10 mM phosphate buffer, pH 7.0) is boiled for 1 min, the radioactivity remains stably attached to the denatured protein. Moreover, whereas the native enzyme is very resistant to the action of various proteases, the denatured protein can be readily degrad-

Crystallographic mapping of β-lactams bound to a d-alanyl-d-alanine peptidase target enzyme☆

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins. Cephalosporin C, the monobactam analog of penicillin G and (2,3)-alpha-methylene benzylpenicillin have been mapped at 2.3 A resolution in the form of acyl-enzyme complexes bound to serine 62. On the basis of the positions of these inhibitors, the binding of a tripeptide substrate for the enzyme, L-lysyl-D-alanyl-D-alanine, has been modeled in the active site. The binding of both inhibitors and substrate is facilitated by hydrogen-bonding interactions with a conserved beta-strand (297-303), which is antiparallel to the beta-lactams acylamide linkage or the substrates peptide bond. The active site is similar to that in beta-lactamases.

[51] Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces

Publisher Summary Strains R39 and R61 are soil isolates. Their designations are arbitrary. In strain R39, the cross-link between the peptide units of the wall peptidoglycan extends from the C-terminal D-alanine of one unit to the amino group at the D-center of meso-diaminopimelic acid of another unit (peptidoglycan of chemotype I). The interpeptide bond is in position to a free carboxyl group. In strain R61, the cross-link extends from a C-terminal D-alanine of a peptide unit to a glycine residue attached to the amino group of LL-diaminopimelic acid of another peptide unit (peptidoglycan of chemotype II). The exocellular DD carboxypeptidases-transpeptidases produced by both strains catalyze hydrolysis, react with β-lactam antibiotics. This chapter explains the assay methods for DD-Carboxypeptidase activity like the standard reaction, chemical estimation of free Alanine, as well as, assay method for β-Lactamase. It also discusses the Excretion of DD-Carboxypeptidase-Transpeptidase and β -Lactamase by Streptomyces R39, Excretion of DD-Carboxypeptidase-Transpeptidase and β-Lactamase by Streptomyces R61, purification of the DD-Carboxypeptidase-Transpeptidase from Streptomyces R39 (for 500 Liters of Culture Fluid), Purification of the DD-Carboxypeptidase-Transpeptidase from Streptomyces R61 (for 400 Liters of Culture Fluid), Physicochemical Properties of DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61, Interaction between DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 and β-Lactam Antibiotics , Titration of DD-Carboxypeptidases-Transpeptidases from Streptornyces R39 and R61 by β-Lactam Antibiotics, Hydrolysis Reactions Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61, Concomitant Hydrolysis and Transfer Reactions Involving Distinct Donor and Acceptor Peptides, Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptonayces R39 and R61, Concomitant Hydrolysis and Transfer Reactions Catalyzed by the DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 and in Which the Same Peptide Acts as Donor and Acceptor and Inhibition of DD-Carboxypeptidases-Transpeptidases from Streptomyces R39 and R61 by β-Lactam Antibiotics in the Presence of Substrates.