A Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo-β-Lactamase.
Abstract
New Delhi metallo-β-lactamase 1 (NDM-1) is a subclass B1 metallo-β-lactamase that exhibits a broad spectrum of activity against β-lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in kcat/Km values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65- and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had kcat/Km values for ceftazidime about 25- and 89-fold higher, respectively, than that of NDM-1.
Submission Details
ID: CWvASvaZ3
Submitter: Shu-Ching Ou
Submission Date: March 20, 2019, 3:27 p.m.
Version: 1
Publication Details
Marcoccia F;Mercuri PS;Galleni M;Celenza G;Amicosante G;Perilli M,Antimicrob Agents Chemother (2018) A Kinetic Study of the Replacement by Site Saturation Mutagenesis of Residue 119 in NDM-1 Metallo-β-Lactamase. PMID:29784851Additional Information
Study Summary
| Number of data points | 299 |
| Proteins | Metallo-beta-lactamase type 2 |
| Unique complexes | 117 |
| Assays/Quantities/Protocols | Experimental Assay: kcat/Km ; Experimental Assay: Km ; Experimental Assay: kcat ; Experimental Assay: MIC |
| Libraries | Variants for NDM-1_Substrate |
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
|---|
Data Distribution
Studies with similar sequences (approximate matches)
Correlation with other assays (exact sequence matches)
Relevant PDB Entries
| Structure ID | Release Date | Resolution | Structure Title |
|---|---|---|---|
| 5XP6 | 2018-03-07 | 0.95 | native structure of NDM-1 crystallized at pH5.5 |
| 5ZGZ | 2018-08-22 | 0.95 | Crystal structure of NDM-1 at pH7.5 (Imidazole) with 1 molecule per asymmetric unit |
| 5ZGY | 2018-08-22 | 0.95 | Crystal structure of NDM-1 at pH7.5 (Bis-Tris) with 1 molecule per asymmetric unit |
| 5ZGX | 2018-08-22 | 0.95 | Crystal structure of NDM-1 at pH7.5 (Succinate) with 1 molecule per asymmetric unit |
| 5ZGW | 2018-08-22 | 0.95 | Crystal structure of NDM-1 at pH7.5 with 1 molecule per asymmetric unit (crystallized at succinate pH5.5 and soaked at succinate pH7.5) |
| 5ZJC | 2019-03-20 | 0.96 | Crystal structure of NDM-1 in complex with D-captopril derivative CY41 |
| 5ZJ8 | 2019-03-20 | 0.96 | Crystal structure of NDM-1 in complex with D-captopril derivative CY32 |
| 5ZJ7 | 2019-03-20 | 0.96 | Crystal structure of NDM-1 in complex with D-captopril derivative CY22 |
| 5ZJ1 | 2019-03-20 | 0.96 | Crystal structure of NDM-1 in complex with D-captopril derivative CYT-14 |
| 5ZGI | 2018-08-22 | 0.98 | Crystal structure of NDM-1 at pH6.5 (Succinate) with 1 molecule per asymmetric unit |
| 5ZIO | 2019-03-20 | 0.98 | Crystal structure of NDM-1 in complex with L-captopril |
| 5ZGE | 2018-08-22 | 1.0 | Crystal structure of NDM-1 at pH5.5 (Bis-Tris) in complex with hydrolyzed ampicillin |
| 6Q2Y | 2019-04-24 | 1.0 | Crystal structure of NDM-1 beta-lactamase in complex with broad spectrum boronic inhibitor cpd3 |
| 5ZH1 | 2018-08-22 | 1.05 | Crystal structure of NDM-1 at pH7.5 (Imidazole) with 2 molecules per asymmetric unit |
| 4HL2 | 2012-12-12 | 1.05 | New Delhi Metallo-beta-Lactamase-1 1.05 A structure Complexed with Hydrolyzed Ampicillin |
| 5ZJ2 | 2019-03-20 | 1.1 | Crystal structure of NDM-1 in complex with D-captopril |
| 6D1I | 2019-04-17 | 1.1 | Crystal structure of NDM-1 complexed with compound 12 |
| 6D1F | 2019-04-17 | 1.15 | Crystal structure of NDM-1 complexed with compound 8 |
| 5ZGR | 2018-08-22 | 1.15 | Crystal structure of NDM-1 at pH7.3 (HEPES) in complex with hydrolyzed ampicillin |
| 6D1G | 2019-04-17 | 1.15 | Crystal structure of NDM-1 complexed with compound 9 |
| 6D1J | 2019-04-17 | 1.15 | Crystal structure of NDM-1 complexed with compound 13 |
| 5ZGV | 2018-08-22 | 1.15 | Crystal structure of NDM-1 at pH8.0 (Tris) with 2 molecules per asymmetric unit |
| 5ZGP | 2018-08-22 | 1.15 | Crystal structure of NDM-1 at pH6.2 (Bis-Tris) in complex with hydrolyzed ampicillin |
| 6MDU | 2019-04-17 | 1.15 | Crystal structure of NDM-1 with compound 7 |
| 6D1E | 2019-04-17 | 1.15 | Crystal structure of NDM-1 complexed with compound 7 |
| 4EYB | 2012-08-15 | 1.16 | Crystal structure of NDM-1 bound to hydrolyzed oxacillin |
| 4EY2 | 2012-08-08 | 1.17 | Crystal structure of NDM-1 bound to hydrolyzed methicillin |
| 5ZGT | 2018-08-22 | 1.2 | Crystal structure of NDM-1 at pH7.5 (HEPES) with 2 molecules per asymmetric unit |
| 5ZGF | 2018-08-22 | 1.2 | Crystal structure of NDM-1 Q123G mutant |
| 6D1K | 2019-04-17 | 1.2 | Crystal structure of NDM-1 complexed with compound 14 |
| 6D1A | 2019-04-17 | 1.25 | Crystal structure of NDM-1 complexed with compound 1 |
| 6D1B | 2019-04-17 | 1.25 | Crystal structure of NDM-1 complexed with compound 2 |
| 6D1H | 2019-04-17 | 1.25 | Crystal structure of NDM-1 complexed with compound 11 |
| 6D1C | 2019-04-17 | 1.25 | Crystal structure of NDM-1 complexed with compound 3 |
| 4RAW | 2014-11-26 | 1.3 | Crystal Structure of New Delhi Metallo-beta-Lactamase-1 Mutant M67V Complexed with Hydrolyzed Ampicillin |
| 4RL0 | 2014-11-19 | 1.3 | Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins |
| 5O2E | 2018-12-26 | 1.3 | Crystal structure of NDM-1 in complex with hydrolyzed cefuroxime - new refinement |
| 4GYQ | 2012-09-26 | 1.35 | Crystal Structure of New Delhi Metallo-beta-Lactamase-1 D223A mutant from Klebsiella pneumoniae |
| 6IBS | 2019-04-24 | 1.37 | Crystal structure of NDM-1 beta-lactamase in complex with boronic inhibitor cpd 6 |
| 6D1D | 2019-04-17 | 1.4 | Crystal structure of NDM-1 complexed with compound 6 |
| 6IBV | 2019-05-15 | 1.4 | Crystal structure of NDM-1 beta-lactamase in complex with broad spectrum boronic inhibitor cpd 1 |
| 5WIG | 2017-10-18 | 1.4 | Structure of New Delhi Metallo-Beta-lactamase 4 (NDM-4) |
| 5N0I | 2017-04-05 | 1.47 | Crystal structure of NDM-1 in complex with beta-mercaptoethanol - new refinement |
| 4EXY | 2012-08-08 | 1.47 | Crystal structure of NDM-1 bound to ethylene glycol |
| 6Q30 | 2019-04-24 | 1.5 | Crystal structure of NDM-1 beta-lactamase in complex with boronic inhibitor cpd 5 |
| 4RAM | 2014-09-24 | 1.5 | Crystal Structure of New Delhi Metallo-beta-Lactamase-1 Mutant M67V Complexed with Hydrolyzed Penicillin G |
| 4HL1 | 2012-12-12 | 1.5 | Crystal Structure of New Delhi Metallo-beta-Lactamase-1, Complexed with Cd and Ampicillin |
| 4H0D | 2012-09-26 | 1.5 | New Delhi Metallo-beta-Lactamase-1 Complexed with Mn from Klebsiella pneumoniae |
| 5ZGQ | 2018-08-22 | 1.5 | Crystal structure of NDM-1 at pH7.5 (Tris-HCl, (NH4)2SO4) in complex with hydrolyzed ampicillin |
| 6RMF | 2019-09-11 | 1.51 | Crystal structure of NDM-1 with VNRX-5133 |
| 5ZGU | 2018-08-22 | 1.55 | Crystal structure of NDM-1 at pH7.0 (HEPES) with 2 molecules per asymmetric unit |
| 5XP9 | 2018-03-07 | 1.55 | Crystal structure of Bismuth bound NDM-1 |
| 6MGY | 2018-10-03 | 1.6 | Crystal Structure of the New Deli Metallo Beta Lactamase Variant 5 from Klebsiella pneumoniae |
| 6C6I | 2018-10-17 | 1.65 | Crystal structure of a chimeric NDM-1 metallo-beta-lactamase harboring the IMP-1 L3 loop |
| 6MGZ | 2018-10-03 | 1.65 | Crystal Structure of the New Deli Metallo Beta Lactamase Variant 4 from Klebsiella pneumoniae |
| 6EFJ | 2019-04-17 | 1.65 | Crystal structure of NDM-1 with compound 9 |
| 5JQJ | 2017-05-10 | 1.67 | Directed evolutionary changes in MBL super family - NDM-1 Round 10 crystal-1 |
| 6CAC | 2018-10-17 | 1.79 | Crystal structure of NDM-1 metallo-beta-lactamase harboring an insertion of a Pro residue in L3 loop |
| 4EYF | 2012-08-08 | 1.8 | Crystal structure of NDM-1 bound to hydrolyzed benzylpenicillin |
| 4GYU | 2012-09-26 | 1.8 | Crystal Structure of New Delhi Metallo-beta-Lactamase-1 A121F mutant from Klebsiella pneumoniae |
| 4EYL | 2012-08-08 | 1.9 | Crystal structure of NDM-1 bound to hydrolyzed meropenem |
| 5N0H | 2017-04-05 | 1.9 | Crystal structure of NDM-1 in complex with hydrolyzed meropenem - new refinement |
| 4U4L | 2014-08-20 | 1.9 | Crystal structure of the metallo-beta-lactamase NDM-1 in complex with a bisthiazolidine inhibitor |
| 3ZR9 | 2011-06-29 | 1.91 | Structure of New Delhi Metallo-Beta-lactamase 1 (NDM-1) |
| 6EX7 | 2017-12-13 | 1.95 | Crystal structure of NDM-1 metallo-beta-lactamase in complex with Cd ions and a hydrolyzed beta-lactam ligand - new refinement |
| 5K4M | 2017-04-26 | 1.98 | Directed evolutionary changes in MBL super family - NDM-1 Round 10 crystal-3 |
| 4HKY | 2013-01-23 | 2.0 | New Delhi Metallo-beta-Lactamase-1, Complexed with Cd and Faropenem |
| 3PG4 | 2011-11-16 | 2.0 | The crystal structure of New Delhi Metallo-beta lactamase (NDM-1) |
| 3RKJ | 2011-05-18 | 2.0 | Crystal Structure of New Delhi Metallo-Beta-Lactamase-1 from Klebsiella pnueumoniae |
| 5O2F | 2018-12-26 | 2.01 | Crystal structure of NDM-1 in complex with hydrolyzed ampicillin - new refinement |
| 4RL2 | 2014-11-19 | 2.01 | Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins |
| 4RM5 | 2014-11-19 | 2.1 | Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins |
| 3SPU | 2011-08-03 | 2.1 | apo NDM-1 Crystal Structure |
| 3SFP | 2011-06-29 | 2.27 | Crystal Structure of the Mono-Zinc-boundform of New Delhi Metallo-beta-Lactamase-1 from Klebsiella pneumoniae |
| 3SBL | 2011-06-29 | 2.31 | Crystal Structure of New Delhi Metal-beta-lactamase-1 from Klebsiella pneumoniae |
| 3RKK | 2011-05-18 | 2.35 | Crystal Structure of New Delhi Metallo-Beta-Lactamase-1 from Klebsiella pneumoniae |
| 4EXS | 2012-08-08 | 2.4 | Crystal structure of NDM-1 bound to L-captopril |
| 4RBS | 2014-11-26 | 2.4 | Crystal Structure of New Delhi Metallo-beta-Lactamase-1 in the Complex with Hydrolyzed Meropenem |
| 3SRX | 2011-07-20 | 2.5 | New Delhi Metallo-beta-Lactamase-1 Complexed with Cd |
| 5A5Z | 2015-07-08 | 2.6 | Approved Drugs Containing Thiols as Inhibitors of Metallo-beta- lactamases: Strategy To Combat Multidrug-Resistant Bacteria |
| 5NBK | 2018-10-03 | 2.6 | NDM-1 metallo-beta-lactamase: a parsimonious interpretation of the diffraction data |
Relevant UniProtKB Entries
| Percent Identity | Matching Chains | Protein | Accession | Entry Name |
|---|---|---|---|---|
| 100.0 | Metallo-beta-lactamase type 2 | C7C422 | BLAN1_KLEPN |