[51] Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces

Frère, Jean‐Marie; Leyh‐Bouille, Mélina; Ghuysen, Jean‐Marie; Nieto, Manuel; Perkins, H. R.

doi:10.1016/s0076-6879(76)45054-1

Cited by 97 publications

(57 citation statements)

References 24 publications

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“…The hydrolysis of Ac2-L-Lys-D-Ala-D-Ala was stopped by heating the solutions in boiling water for 1 min and the amount of released D-Ala measured enzymatically (D-amino acid oxidase procedure) as described [19]. Alternatively, the amount of [ ''C]Ac2-~-Lys-~-Ala released from [ ''C]Ac2-L-Lys-D-Ala-D-Ala was separated from the residual substrate by paper electrophoresis and estimated as described [15].…”

Section: Im-peptidase Activitymentioning

confidence: 99%

The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61

Varetto

Frère

Nguyen-Distèche

et al. 1987

European Journal of Biochemistry

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Titration of the active-site serine DD-peptidase of Streptomyces R61 shows that formation of acyl enzyme during hydrolysis of the substrate Ac,-~-Lys-~-Aia-~-Ala and enzyme inactivation by the p-lactam compounds benzylpenicillin, N-acetylampicillin and ampicillin relies on the acidic form of an enzyme's group of pK z 9.5. It is proposed that protonation of a lysine &-amino group facilitates initial binding by charge pairing with the free carboxylate of the substrate and the 8-lactam molecules. Lowering the pH from 7 to 5 has no effect on the secondorder rate constant of enzyme acylation by benzylpenicillin and N-acetylampicillin but results in a decreased rate constant of acylation by ampicillin and Ac,-~-Lys-~-Ala-~-Ala. Protonation of the side-chain amino group of ampicillin and a decreased efficacy of the initial binding of the peptide to the enzyme seem to be responsible for the observed effects. Whatever the molecule bound to the enzyme, there is no sign for the active involvement of an enzyme's histidine residue of pK 6.5 -7.0 in the hydrolysis pathway.Two families of bacterial enzymes specifically interact with p-lactam compounds. The p-lactamases, which hydrolyse the p-lactam ring, can play an important role in resistance. The DD-peptidases, which participate in the last stages of wall peptidoglycan metabolism, are the primary targets of these antibiotics [l, 21. Many of the /3-lactamases (classes A and C) and DD-peptidases characterized so far are serine enzymes and react with p-lactam compounds according to reaction (1).(1)where E = enzyme, D = p-lactam, k + and k , , = first-order rate constants, K = dissociation constant of E . D (Michaelis complex) and E -D* = serine ester-linked acyl enzyme [3]. Most often, the value of k , , is high or very high with the j-lactamases, and so low with the DD-peptidases that a slight stoichiometric excess of p-lactam compound causes longlasting immobilization of the protein at the level of the acyl enzyme and these latter enzymes behave as penicillin-binding and C, the active-site serine is followed by an Xaa-Xaa-Lys sequence (which suggests that this lysine probably plays an important role), little is known concerning the enzymes' functional groups involved in substrate binding and catalysis. where HY is H 2 0 or a suitable R-NH2 peptide or amino acid. The goal of the present work was to obtain further information on the possible functional groups involved in catalysis and penicillin binding by the Streptomyces R61 DD-peptidase. When this study was initiated, it was known [2] that at pH 7.0 and 37°C (unless otherwise stated) (a) the K , k + , and k+, values for the interaction with benzylpenicillin were 13 mM (25"C), 180s-I (25°C) and 1 . 4~1 0 -~s -' ; (b) the corresponding values for the interaction with ampicillin were 7.2 mM, 0.77 s-l and 1.4 x s-'; (c) the K,,, and k,,, values for the best substrate Ac,-~-Lys-~-Ala-~-Ala were about 10 mM and 50 S K I , respectively and (d) the activity of the enzyme decreased with increasing ionic strength [15]. MATERIALS AND...

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Section: Im-peptidase Activitymentioning

confidence: 99%

The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61

Varetto

Frère

Nguyen-Distèche

et al. 1987

European Journal of Biochemistry

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“…by the D-amino acid oxidase method (6). In the case of the K15 enzyme, the assay mixture also contained 10 mM glycylglycine.…”

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confidence: 99%

CENTA as a Chromogenic Substrate for Studying β-Lactamases

Bebrone

Moali

Mahy

et al. 2001

Antimicrob Agents Chemother

101

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CENTA, a chromogenic cephalosporin, is readily hydrolyzed by ␤-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of ␤-lactamaseproducing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.Nitrocefin and, to a lesser extent, PADAC have been used as chromogenic substrates of ␤-lactamases. Such substrates, whose hydrolysis can be directly monitored in the visible wavelength range, are of particular interest for the kinetic characterization of ␤-lactamases. Nitrocefin has, for instance, been widely used as a reporter substrate in the study of the inactivation of ␤-lactamases or of their interactions with poor substrates (3). It also allows the rapid identification of active fractions during ␤-lactamase purification. However, the price of nitrocefin has recently been increased significantly, and PA-DAC is no longer commercially available. Synthesis of compounds is also rather tedious. It is thus surprising that a third chromogenic cephalosporin, CENTA ( Fig. 1), which can be prepared from the commercially available drug cephalothin, has not received more attention, although it was shown to be sensitive to many ␤-lactamases (11). In the study described in this report, we determined the kinetic parameters characterizing the interactions between CENTA and a representative set of ␤-lactamases and some penicillin-binding proteins (PBPs). Although CENTA cannot be used for the direct detection of ␤-lactamase-producing colonies on agar plates, it still represents an interesting alternative to nitrocefin for the kinetic characterization of ␤-lactamases.CENTA was prepared as follows: 3-carboxyl-4-nitrothiophenol (TNB) was obtained by dissolving 5.05 mmol (2 g) of 5,5Ј-dithio-bis-(2-nitrobenzoic acid) in 100 ml of an aqueous solution of 0.5 M Tris base, and the pH was adjusted to 8.0 by addition of 6 M HCl. Dithiothreitol (7.1 mmol, 1.1 g) was added, and the solution turned orange-red. The mixture was stirred for 10 min at 22°C and extracted six times with 25 ml of ethyl acetate before being acidified to pH 1.5 by addition of 6 M HCl. The residual ethyl acetate was eliminated by bubbling nitrogen through the solution, which was thereafter left overnight at 4°C. The precipitate (TNB) was collected by filtration, washed, and dried. The sodium salt of cephalothin (1 g, 2.4 mmol) and 1 equivalent of TNB (478 mg) were dissolved in 19 ml of H 2 O, and the pH was adjusted to 7.0 with 1 M NaOH. The solution was stirred for 6 h at 65°C. The cooled solution was extracted with 10 ml of ethyl acetate, acidified to pH 2.0 with 1 M HCl, and extracted three times with 15 ml of ethyl acetate. The organic phase was washed three times with 15 ml of water, dried over MgSO 4 , and evaporated to dryness in vacuo. The sodium salt of CENTA was obtained by dissolving the dry residue in 25 ml of water containing 1 equivalent of NaHCO 3 , and the solution was freeze-dried,...

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“…22 Samples for assay of DD-carboxypeptidase activity consisted of 10 ml of exocellular DD-carboxypeptidase 64-575 II (50 mM) from Saccharopolyspora erythraea 64-575 II (Polish Collection of Microorganism, Wroclaw, accession number B/00018), 9,23 10 ml of the substrate solution containing 2.98 mg ml À1 N a ,N e -diacetyl-L-lysyl-D-alanyl-D-alanine in 0.1 M phosphate buffer, pH 8.0, and 20 ml of 0.1M phosphate buffer, pH 8.0. Standard sample contained 20 ml of D-alanine in distilled water.…”

Section: Assay Of Dd-carboxypeptidase Activitymentioning

confidence: 99%

A novel isoquinoline alkaloid, DD-carboxypeptidase inhibitor, with antibacterial activity isolated from Streptomyces sp. 8812. Part I: Taxonomy, fermentation, isolation and biological activities

2009

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A novel isoquinoline alkaloid of molecular formula C 10 H 9 NO 4 , labeled JS-1, was isolated from the culture broth of Streptomyces sp. 8812. It was purified by acetone protein precipitation from the culture supernatant, followed by anion exchange and C18 RP HPLC columns. JS-1 is an inhibitor of exocellular DD-carboxypeptidases/transpeptidases (DD-peptidases) 64-575 II from Saccharopolyspora erythraea 64-575 II, and R39 from Actinomadura R39. JS-1 exhibits activity against Gram-negative bacteria, such as Bordetella bronchiseptica, Stenotrophomonas maltophilia, Proteus vulgaris, P. mirabilis, Burkholderia cepacia and Acinetobacter baumanii, with MIC values 10-160 lg ml À1 , and against Gram-positive bacteria, such as Staphylococcus aureus, with MIC values 40-206 lg ml À1 .

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[51] Exocellular dd-carboxypeptidases-transpeptidases from Streptomyces

Cited by 97 publications

References 24 publications

The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61

The pH dependence of the active‐site serine DD‐peptidase of Streptomyces R61

CENTA as a Chromogenic Substrate for Studying β-Lactamases

A novel isoquinoline alkaloid, DD-carboxypeptidase inhibitor, with antibacterial activity isolated from Streptomyces sp. 8812. Part I: Taxonomy, fermentation, isolation and biological activities

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