Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1
| Autor: | M. Tabish Rehman, Asad U. Khan |
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| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular 0301 basic medicine Circular dichroism 030106 microbiology Mutant Gene Expression beta-Lactams Protein Structure Secondary beta-Lactamases Structure-Activity Relationship 03 medical and health sciences Mechanisms of Resistance Enterobacter cloacae Enzyme Stability Escherichia coli Pharmacology (medical) Enzyme kinetics Cloning Molecular Pharmacology Alanine biology Chemistry Hydrolysis Tryptophan Active site Hydrogen Bonding biology.organism_classification Enterobacteriaceae Recombinant Proteins Anti-Bacterial Agents Protein Structure Tertiary Kinetics Infectious Diseases Biochemistry Mutation Biocatalysis biology.protein Thermodynamics Hydrophobic and Hydrophilic Interactions |
| Zdroj: | Antimicrobial Agents and Chemotherapy. 60:356-360 |
| ISSN: | 1098-6596 0066-4804 |
| DOI: | 10.1128/aac.01194-15 |
| Popis: | New Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze β-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 Δ 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 α-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. |
| Databáze: | OpenAIRE |
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