Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

Abstract

New Delhi metallo-␤-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze ␤-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned bla NDM-1 from an Enterobacter cloacae clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4-to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the K m was reduced, while the k cat was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ⌬G D o and T m of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the ␣-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.

The mechanism of NDM-1-catalyzed carbapenem hydrolysis is distinct from that of penicillin or cephalosporin hydrolysis

“…We noted that the ratio of Δ 2 (EI 1 ) to Δ 1 (EI 2 or EP) was 1:5 in imipenem-bound structures but 3:2 in meropenem-bound structures (Tables 1 , 2 ), which probably means that there are inconsistent stereoselective advantages between the two carbapenem substrates. More Δ 1 present in hydrolyzed imipenem may hint at a faster rate of pyrroline tautomerization, in agreement with the larger k cat value for imipenem than that of meropenem in NDM-1-mediated hydrolysis 13 , 14 , 41 . It is possible that the formimidamide group in imipenem is more favorable for intermediate protonation, as it provides a more hydrophilic milieu for a bulky solvent molecule to approach, while the relatively hydrophobic pyrrolidine ring in meropenem is somewhat unfavorable.…”

Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health

“…Tn3000 transposon has been reported to be responsible for bla NDM-1 dissemination among enterobactericeae [ 65 ]. A non-active site residue Trp 93 is found to play role in maintaining the structural integrity of NDM-1, although not being directly involved in recognition and catalysis [ 66 ]. Recently, a new plasmid type IncX3 is reported to be responsible for making the spread of NDM gene more effective [ 67 ], for example in China IncX3 and InA/C plasmids were reported to be responsible for spread of bla NDM genes [ 68 ].…”

Biogenesis of ZnO nanoparticles using Pandanus odorifer leaf extract: anticancer and antimicrobial activities

“…21 The emergence of a new persistent bacterial strain is the direct consequence of the non-judicial use of antibiotics. 22 The risk of infection with pathogenic strains is increasing due to the lack of proper medication systems and sterilization techniques and the improper handling and treatment of hazardous materials. 23 For example, infections by Shigella exneri cause 1.5 million deaths annually due to the contamination of food and drinks.…”