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SUBSTITUTION L209F IN NDM-1 METALLO-β-LACTAMASE DRASTICALLY REDUCES ENZYME ACTIVITY

VERSUS Contact information: Francesca Marcoccia F. Marcoccia, C. Bottoni, A. Sabatini, M. Colapietro, G. Celenza, P. Bellio, G. Amicosante, M. Perilli University of L’Aquila [email protected] Department of Biotechnological and Applied Clinical Sciences , University of L'Aquila, L'Aquila, Italy

Introduction Results E. coli BL21 E. coli E. coli BL21 (DE3)/ NDM-1 is a metallo-β-lactamase belonging to subclass B1. It confers Β-Lactams (DE3)/ BL21(DE3)/ pET-24a resistance to one of the most potent class of , known as L209F carbapenems. NDM-1 gene have been rapidly spreading via complex NDM-1 NDM-1 mobile genetic elements among many different types of bacteria (1). >64 0.25 0.5 Especially, NDM-1 has been found in Escherichia coli, the most common bacterium in the human population, which is the cause of common and >64 <0.0625 <0.0625 frequent infections. Moreover the NDM-1 enzyme has been found also in Figure 1: Thermal stability of NDM-1L209F in comparison with that of the NDM-1 0.125 <0.0625 <0.0625 other bacterial species (2). To date do not exist antibiotics and inhibitors enzyme >256 4 <0.0625 able to inactivate MBLs. For these reasons structural studies on NDM-1, as The first approach to study the effect of the mutation in position 209 was to test >256 1 <0.0625 other MBLs enzymes, are needed. the stability of the enzyme at 30°C and 40°C up to 60 minutes. As shown from the >256 16 <0.0625 graph the mutant NDM-1L209F maintains the same stability of the wild-type Aim of the study enzyme NDM-1. A decrease of residual activity of about 20% and 35% at 30°C 16 8 <0.125 In this study a non active site residue of leucine at position 209 is and 40°C was observed, respectively, for both enzymes. replaced by a phenilalanine residue. 32 <0.0625 <0.0625 Loracarbef >128 16 <0.0625 Why position 209? Leucin 209 is far from the active site and does not interact with the 128 16 0.5 substrate or zinc ions. However L209 directly interacts with cystein Moxalactam 64 0.25 <0.125 208, a conserved residue in B1 MBLs which coordinate Zn2. Benzilpenicillin >512 64 0.5 Furthermore L209 shapes an hydrogen bond with a residue of tyrosine at position 229 (3). This residue is conserved in all MBLs of class B2 >512 32 0.5 L209F and B3, but not in class B1, which presented a residue of triptophane Figure 2: Fluorescence emission spectra of NDM-1 (pink line), NDM-1 . (red line). In green the emission spectrum of the buffer Hepes with 20µM ZnCl2 Table 2: Pattern of β-lactam resistance mediated by NDM-1 instead a residue of tyrosine. The substitution with a residue of L209F and NDM-1 in E. coli BL21(DE3) in comparison with the phenylalanine has been planned on the basis of the amino acid Fluorescence spectra was carried out to study the conformation of NDM-1 and L209F pattern in E. coli BL21 (DE3)/pET-24a sequence alignment with IMP-1 (MBL B1). NDM-1 by following the emission of tryptophan and tyrosine residues. The experiments were performed in a Perkin-Elmer LS50B (Perkin-Elmer, Monza, As observed for catalytic efficiencies the mutation L209F Italy) by using 80 nM of each enzyme in Hepes at a 280 nm excitation and at 300- produces a drastic reduction of in vivo resistance level. NDM-1 modelling was performed with 460 nm emission wavelengths. The results have shown that there is a difference Especially MIC experiments have pointed out an inability for Swiss pdb Viewer using 3Q6X.pdb file. L209F Zinc ions are shown as blue spheres. between the folding of the mutant and the wild-type enzyme. The higher peak NDM-1 to growth in the presence of Carbapenems. observed in NDM-1L209F reveals that the mutant has a more packed conformation than NDM-1 enzyme. Conclusions The introduction of phenylalanine in position 209 drastically reduces the activity of NDM-1 especially towards Methods Carbapenems. The existence of an aromatic group as NDM-1L209F was generated by site-directed mutagenesis using the overlap phenylalanine could disturb the interaction with the L209F extension method (4). NDM-1 and NDM-1 were generated without the conserved residue Y229 and with the catalytic residue C208 signal peptide due to their stability and were inserted both into pET24(a) by shifting the position of these amino acid residues or by vector and transferred in E. coli HB101. E. coli BL21 (DE3) were used as changing the Van der Waals forces. This study highlighted recipient for transformation assay. The phenotypic profile has been the importance of L209 not only for the catalytic activity of 5 characterized by microdilution method using a bacterial inoculum of 5 x 10 NDM-1 enzyme but also for its structure integrity. CFU/mL according to Clinical and Laboratory Standards Institute (CLSI) Table 1: Kinetic parameters of the NDM-1 and NDM-1L209F, enzymes towards some performance standards (5). NDM-1 and NDM-1L209F enzymes were purified β-lactams References by two chromatographic steps from 2L of a culture of E.coli BL21 DE3 NDM-1 and NDM-1L209F were analysed against different β-lactam antibiotics. As 1. Kumarasamy KK et al.Lancet Infect Dis 2010;10:597-602 grown at 37°C in an orbital shaker (180 rpm). Each culture was grown to displayed from the kinetic experiments, the mutant shows a radical decrease of kcat 2. Deshpande P et al. J Assoc Physicians India 2010;58:147-9 achieve an A600 of approximately 0.5 OD and 0.4 mM IPTG (isopropyl-ß- values for Carbapenems (Imipenem, Meropenem and Biapenem) and for 3.Chen J, et al. PlosOne 2013; 8: 1-8 thiogalactoside) was added. Steady-state kinetic experiments were performed (Loracarbef and Cefotaxime) while maintaining the Km values 4.Steffan NH et al. Gene 1989 ; 77 :51-9. following the hydrolysis of each substrate at 25°C in 20 mM Hepes buffer similar to the wild-type enzyme. With the exception of (Carbenicillin 5. Clinical and Laboratory Standards Instituts. Document and Benzilpenicillin) for which there was not observed hydrolysis. M7-A7, 26 (2). CLSI, Wayne, PA, USA, 2006. (pH 7.0) containing 0.2 M KCl and 20 µM of ZnCl2.