Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1 I35T and NDM-1 I35S enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. The k cat , K m , and k cat /K m values calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, the k cat /K m of NDM-1 I35S for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1 I35T and NDM-1 I35S enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of ␣-helix content in the mutants.
The NDM-1 metallo--lactamase (MBL) was first described in a urinary Klebsiella pneumoniae isolate recovered from a Swedish patient who traveled to New Delhi and who had received medical care in India (1). This is the most recent MBL to have widely spread around the world among enterobacterial strains, Pseudomonas aeruginosa (2), Acinetobacter baumannii (3), Morganella morganii (4), Alcaligenes faecalis, Vibrio cholerae, and Stenotrophomonas maltophilia (5, 6). NDM-1-producing bacteria have been recovered from many infection sites as hospital-acquired and community-acquired infections but also in environmental samples (7). Since its finding, 12 NDM variants have been identified (http://www.lahey.org/Studies/). The factor that has influenced the wide geographic spread of bla NDM-1 gene is its localization on complex plasmids that mediate the transfer of this resistant determinant under the selective pressure of antibiotic therapy (8, 9). Metallo--lactamases show a broad-spectrum substrate profile; they are resistant to classical -lactamase inhibitors and hydrolyze carbapenems very efficiently. MBLs require one or two zinc ions to catalyze the hydrolysis of -lactams. It is commonly suggested that the zinc ion acts as a Lewis acid to stabilize the transient tetrahedral intermediate formed by the nucleophilic attachment of a hydroxide ion to the carboxyl group of the -lactam ring (10). NDM-1 enzyme shows a great ability to hydrolyze all -lactam antibiotics (11). Several crystal structures of NDM-1 have been solved, and the enzyme displays the typical ␣/␣ fold of MBLs (12,13,14). NDM-1 belongs to subclass B1, and it contains a binuclear Zn active site surrounded by several loops responsible for substrate binding and specificity (13). Several studies pointed out the attention focused on the L3 loop, which could be involved in the hydrophobic contacts with substrates through the presence of an aromatic residue. Moreover, its great flexibility makes the loop able to drive substrate into the active site. The role of the L3 loop has been studied in such subclass B1 MBLs as IMP-1 and VIM-2 (15), but no experimental data are available for the NDM-1 enzyme. In comparison to the IMP and VIM variants, NDM-1 has a longer N terminus which forms two extra strands that pack on the L3 loop through an isoleucin...
bSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo--lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.
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