[en] The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal P-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-p-lactamase Bell. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind Bell, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-beta-lactamases to inactive monobactams and give some clues for inhibitor design. (c) 2007 Elsevier Ltd. All rights reserved.
Disciplines :
Chemistry
Author, co-author :
Poeylaut-Palena, Andres A
Tomatis, Pablo E
Karsisiotis, Andreas I
Damblon, Christian ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique structurale
Mata, Ernesto G
Vila, Alejandro J
Language :
English
Title :
A minimalistic approach to identify substrate binding features in B1 Metallo-beta-lactamases
Dal Peraro M., Vila A.J., and Carloni P. Proteins 54 (2004) 412
Spencer J., Read J., Sessions R.B., Howell S., Blackburn G.M., and Gamblin S.J. J. Am. Chem. Soc. 127 (2005) 14439
Bacillus cereus MβL BcII was obtained according to already published methods by Orellano et al. For UV spectroscopy, hydrolysis assays were carried out in 10 mM KH2PO4, 200 mM NaCl, 20 μM ZnSO4, 50 μg/mL BSA, pH 7.5, at 303 K. For aztreonam, hydrolysis was also tested in 10 mM Hepes, 200 mM NaCl, 20 μM ZnSO4, and BSA 50 μg/mL, pH 7.5. Spectral parameters are: aztreonam λmax = 318 nm, Δε = 660 M-1 cm-1; azetidinone (5) λmax = 195 nm. The spectral features of 5 were determined by following its alkaline hydrolysis. For 1H NMR spectroscopy, hydrolysis assays were carried out in a D2O buffer containing KH2PO4 10 mM, NaCl 200 mM, BSA 50 μg/mL, pH 7.5, at 303 K. The samples were incubated overnight with 10 μM BcII. The NMR experiments were recorded in a Bruker Avance-300 spectrometer.
Orellano E.G., Girardini J.E., Cricco J.A., Ceccarelli E.A., and Vila A.J. Biochemistry 37 (1998) 10173
Fluorescence quenching experiments were carried on an Applied Photophysics SX.18-MVR stopped-flow apparatus. The method was the same as already reported by Rasia et al. Final enzyme concentration was 20 μM in 100 mM Hepes, 200 mM NaCl, pH 7.5, at 288 K. Aztreonam concentrations were in the μM range. The highest employed azetidinone concentration was 150 mM.
For NMR experiments, labeled BcII was obtained as follows: an 8 h LB (150 μg/mL ampicillin and 35 μg/mL cloramphenicol) culture of the transformed cells was pelleted and inoculated into a 100 mL M9 medium supplemented with 4 g/L glucose, 1.2 g/L (NH4)2SO4, 1 mM MgSO4, 10 μM CaCl2, 150 μg/mL ampicillin, and 35 μg/mL cloramphenicol. Cells were grown at 37 °C overnight, pelleted, and resuspended in 2 L of the same fresh medium, but 15N (NH4)2SO4 was used. Cells were grown for 4-5 h at 37 °C until OD600nm = 0.7 was reached. Expression was induced by adding 1 mM isopropyl-thiogalactoside and 100 μM ZnSO4. Cells were cultured for additional 4-5 h until saturation (OD600nm = 1.8) was reached. Cells were harvested and BcII was purified as previously reported by Orellano et al. NMR experiments were carried out at 308 K in Avance II 600 Bruker spectrometer using buffer MES 100 mM, NaCl 200 mM, ZnSO4 200 μM, pH 6.4. BcII samples were in the 0.25-1.1 mM concentration range. Azetidinone (5) and aztreonam (4) stock solutions were 1 M in the same buffer but without added zinc(II).
Mollard C., Moali C., Papamicael C., Damblon C., Vessilier S., Amicosante G., Schofield C.J., Galleni M., Frere J.-M., and Roberts G.C.K. J. Biol. Chem. 276 (2001) 45015
