Overproduction and properties of the mannuronate alginate lyase AlxMB

peer reviewedIn previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 mu g per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked ALxM(A) and unnicked ALxM(B) alginate lyases have identical alginate-degrading activities at high salt concentrations

Site-Directed Mutagenesis of the Streptomyces R61 Dd-Peptidase. Catalytic Function of the Conserved Residues around the Active Site and a Comparison with Class-a and Class-C Beta-Lactamases

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65 → Arg mutant with the peptide and thiol ester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20 000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe358 → Leu, Tyr90 → Asn, Thr101 → Asn, Phe164 → Ala, Asp225 → Glu and Asp225 → Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164 → Ala mutant was significantly more unstable than the wild-type enzyme

Crystal structure of the Actinomadura R39 DD-peptidase reveals new domains in penicillin-binding proteins.

Actinomadura sp. R39 produces an exocellular DD-peptidase/penicillin-binding protein (PBP) whose primary structure is similar to that of Escherichia coli PBP4. It is characterized by a high beta-lactam-binding activity (second order rate constant for the acylation of the active site serine by benzylpenicillin: k2/K = 300 mm(-1) s(-1)). The crystal structure of the DD-peptidase from Actinomadura R39 was solved at a resolution of 1.8 angstroms by single anomalous dispersion at the cobalt resonance wavelength. The structure is composed of three domains: a penicillin-binding domain similar to the penicillin-binding domain of E. coli PBP5 and two domains of unknown function. In most multimodular PBPs, additional domains are generally located at the C or N termini of the penicillin-binding domain. In R39, the other two domains are inserted in the penicillin-binding domain, between the SXXK and SXN motifs, in a manner similar to "Matryoshka dolls." One of these domains is composed of a five-stranded beta-sheet with two helices on one side, and the other domain is a double three-stranded beta-sheet inserted in the previous domain. Additionally, the 2.4-angstroms structure of the acyl-enzyme complex of R39 with nitrocefin reveals the absence of active site conformational change upon binding the beta-lactams

Interactions between non-classical β-lactam compounds and the β-lactamases of Actinomadura R39 and Streptomyces albus G

Streptomyces albus G secretes a Zn2+-containing D-alanyl-D-alanine peptidase. Streptomyces R61 and Actinomadura R39 secrete D-alanyl-D-alanine-cleaving serine peptidases. The effect of non-classical beta-lactam antibiotics on these three model enzymes has been studied. Mecillinam, cefoxitin, quinacillin, quinacillin sulphone, clavulanate and N-formimidoylthienamycin have no effect on the Zn2+-containing enzyme. 6-Amino-penicillanic acid slowly inactivates this enzyme and 7-aminocephalosporanic acid behaves as a reversible inhibitor. Cefoxitin and N-formimidoylthienamycin are potent anti-bacterial agents; they effectively inactivate the serine R39 enzyme and, to a lesser extent, the serine R61 enzyme. All the other beta-lactam compounds tested, including mecillinam, are slow inactivators of these serine enzymes. The intermediates formed between 6-aminopenicillanic acid and the R61 and R39 enzymes are long- and short-lived respectively, whereas those formed between 7-aminocephalosporanic acid and the same R61 and R39 enzymes are short- and long-lived respectively. Breakdown of the short-lived intermediates thus obtained gives rise to several ninhydrin-positive degradation products. The intermediates formed between clavulanate and the serine enzymes are long-lived. With the R39 enzyme, the inactivated complex formed in a first step undergoes subsequent monomolecular rearrangement to give rise to a second species exhibiting a high absorbance at 273 nm

The Division and Cell Wall Gene Cluster of Enterococcus Hirae S185

A chromosomal 10355-bp segment of Enterococcus hirae S185 contains nine orfs which occur in the same order as the MraW-, FtsL-, PBP3-, MraY-, MurD-, MurG-, FtsQ-, FtsA- and FtsZ-encoding genes of the division and cell wall clusters of Escherichia coli and Bacillus subtilis. The E. hirae DNA segment lacks the genes which in E. coli encode the ligases Ddl, MurC, MurE and MurF and the integral membrane protein FtsW. The encoded E. hirae and E. coli proteins share 25% to 50% identity except FtsL and FtsQ (approximately = 14% identity)

Dual substrate specificity of Bacillus subtilis PBP4a

Bacterial DD-peptidases are the targets of the β-lactam antibiotics. The sharp increase in bacterial resistance toward these antibiotics in recent years has stimulated the search for non- β-lactam alternatives. The substrates of DD-peptidases are elements of peptidoglycan from bacterial cell walls. Attempts to base DD-peptidase inhibitor design on peptidoglycan structure, however, have not been particularly successful to date because the specific substrates for most of these enzymes are unknown. It is known, however, that the preferred substrates of low-molecular mass (LMM) class B and C DD-peptidases contain the free N-terminus of the relevant peptidoglycan. Two very similar LMMC enzymes, for example, the Actinomadura R39 DD-peptidase and Bacillus subtilis PBP4a, recognize a D-α-aminopimelyl terminus. The peptidoglycan of B. subtilis in the vegetative stage, however, has the N-terminal D-α-aminopimelyl carboxylic acid amidated. The question is, therefore, whether the DD-peptidases of B. subtilis are separately specific to carboxylate or carboxamide or have dual specificity. This paper describes an investigation of this issue with B. subtilis PBP4a. This enzyme was indeed found to have a dual specificity for peptide substrates, both in the acyl donor and in the acyl acceptor sites. In contrast, the R39 DD-peptidase, from an organism in which the peptidoglycan is not amidated, has a strong preference for a terminal carboxylate. It was also found that acyl acceptors, reacting with acyl−enzyme intermediates, were preferentially D-amino acid amides for PBP4a and the corresponding amino acids for the R39 DD-peptidase. Examination of the relevant crystal structures, aided by molecular modeling, suggested that the expansion of specificity in PBP4a accompanies a change of Arg351 in the R39 enzyme and most LMMC DD-peptidases to histidine in PBP4a and its orthologs in other Bacillus sp. This histidine, in neutral form at pH 7, appeared to be able to favorably interact with both carboxylate and carboxamide termini of substrates, in agreement with the kinetic data. It may still be possible, in specific cases, to combat bacteria with new antibiotics based on particular elements of their peptidoglycan structure

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class a penicillin-binding protein

soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli (14)C(-)labeled lipid 11 precursor. As a DD-peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k(2)/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs

Solubilisation and binding characteristics of a recombinant beta(2)-adrenergic receptor expressed in the membrane of Escherichia coli for the multianalyte detection of beta-agonists and antagonists residues in food-producing animals

The number of substances with beta-agonistic activity, illegally introduced in meat production or in sports doping as anabolic or beta-blocking agents is increasing. Analytical methods suited for their multianalyte detection are thus necessary. In this perspective, receptor assays were developed. The research activities undertaken in this study describe the solubilisation of a recombinant human beta(2)-adrenergic receptor produced in the inner membrane of genetically modified Escherichia coli, using the detergent n-dodecyl-beta-D-maltoside. Its potential to detect the presence of beta-agonists or beta-blockers in biological samples was evaluated. The solubilised beta(2)-adrenergic receptor retained its binding affinity in a radio-receptor assay based on the competition for the binding to receptors between a ligand (beta-agonist or antagonist) and the radioligand [I-125]iodocyanopindolol. The IC50 values ranged from 5 +/- x 10(-8) M (clenbuterol) to 8 +/- 2 x 10(-6) M (isoxsuprine) for the beta-agonists tested and from 1.5 +/- 0.2 x 10(-10) M (carazolol) to 1.2 +/- 0.2 x 10(-5) M (metoprolol) for the beta-blockers tested. It was shown to have a lower limit of detection than a radio-receptor assay using the solubilised beta(2)-adrenoceptor expressed in a mammalian cell line. The solubilised recombinant human beta(2)-adrenoreceptor expressed in E. coli would be a useful tool to develop non radioactive multianalyte screening methods. (c) 2007 Elsevier B.V. All rights reserved