Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability.


Abstract

OBJECTIVES: Metallo-β-lactamase (MBL)-based resistance is a threat to the use of most β-lactam antibiotics. Multiple variants of the New Delhi MBL (NDM) have recently been reported. Previous reports indicate that the substitutions affect NDM activity despite being located outside the active site. This study compares the biochemical properties of seven clinically reported NDM variants. METHODS: NDM variants were generated by site-directed mutagenesis; recombinant proteins were purified to near homogeneity. Thermal stability and secondary structures of the variants were investigated using differential scanning fluorimetry and circular dichroism; kinetic parameters and MIC values were investigated for representative carbapenem, cephalosporin and penicillin substrates. RESULTS: The substitutions did not affect the overall folds of the NDM variants, within limits of detection; however, differences in thermal stabilities were observed. NDM-8 was the most stable variant with a melting temperature of 72°C compared with 60°C for NDM-1. In contrast to some previous studies, kcat/KM values were similar for carbapenem and penicillin substrates for NDM variants, but differences in kinetics were observed for cephalosporin substrates. Apparent substrate inhibition was observed with nitrocefin for variants containing the M154L substitution. In all cases, cefoxitin and ceftazidime were poorly hydrolysed with kcat/KM values <1 s(-1) μM(-1). CONCLUSIONS: These results do not define major differences in the catalytic efficiencies of the studied NDM variants and carbapenem or penicillin substrates. Differences in the kinetics of cephalosporin hydrolysis were observed. The results do reveal that the clinically observed substitutions can make substantial differences in thermodynamic stability, suggesting that this may be a factor in MBL evolution.

Submission Details

ID: itZWKUE8

Submitter: Paulie Dang

Submission Date: Sept. 24, 2019, 10:10 a.m.

Version: 1

Publication Details
Makena A;Brem J;Pfeffer I;Geffen RE;Wilkins SE;Tarhonskaya H;Flashman E;Phee LM;Wareham DW;Schofield CJ,J Antimicrob Chemother (2015) Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability. PMID:25324420
Additional Information

Additional Ki data for Nitrocefin: For NDM-1, NDM-3, NDM-4, NDM-5, NDM-6, NDM-7, and NDM-8: Ki (μM) is not reported (NR), NR, 102 ± 32, 139 ± 28, NR, 79 ± 18, and 146 ± 47, respectively. MIC Assay Data with the ISAba125 promoter were chosen for input into ProtaBank. For MIC Assay Absolute Data with > symbols the next higher multiplier was used to calculate the MIC Relative data.

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
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
6TWT 2020-05-13 0.95 Crystal structure of N-terminally truncated NDM-1 metallo-beta-lactamase
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
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)
5ZGX 2018-08-22 0.95 Crystal structure of NDM-1 at pH7.5 (Succinate) 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
5ZJ8 2019-03-20 0.96 Crystal structure of NDM-1 in complex with D-captopril derivative CY32
5ZJ1 2019-03-20 0.96 Crystal structure of NDM-1 in complex with D-captopril derivative CYT-14
5ZJC 2019-03-20 0.96 Crystal structure of NDM-1 in complex with D-captopril derivative CY41
5ZJ7 2019-03-20 0.96 Crystal structure of NDM-1 in complex with D-captopril derivative CY22
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
6V1M 2020-03-25 1.05 Structure of NDM-1 bound to QPX7728 at 1.05 A
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
6D1I 2019-04-17 1.1 Crystal structure of NDM-1 complexed with compound 12
5ZJ2 2019-03-20 1.1 Crystal structure of NDM-1 in complex with D-captopril
6D1G 2019-04-17 1.15 Crystal structure of NDM-1 complexed with compound 9
6MDU 2019-04-17 1.15 Crystal structure of NDM-1 with compound 7
5ZGV 2018-08-22 1.15 Crystal structure of NDM-1 at pH8.0 (Tris) with 2 molecules per asymmetric unit
6D1J 2019-04-17 1.15 Crystal structure of NDM-1 complexed with compound 13
6D1E 2019-04-17 1.15 Crystal structure of NDM-1 complexed with compound 7
5ZGR 2018-08-22 1.15 Crystal structure of NDM-1 at pH7.3 (HEPES) in complex with hydrolyzed ampicillin
6D1F 2019-04-17 1.15 Crystal structure of NDM-1 complexed with compound 8
5ZGP 2018-08-22 1.15 Crystal structure of NDM-1 at pH6.2 (Bis-Tris) in complex with hydrolyzed ampicillin
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
6D1K 2019-04-17 1.2 Crystal structure of NDM-1 complexed with compound 14
5ZGF 2018-08-22 1.2 Crystal structure of NDM-1 Q123G mutant
5ZGT 2018-08-22 1.2 Crystal structure of NDM-1 at pH7.5 (HEPES) with 2 molecules per asymmetric unit
6LHE 2020-09-16 1.21 Crystal Structure of Gold-bound NDM-1
6D1H 2019-04-17 1.25 Crystal structure of NDM-1 complexed with compound 11
6D1B 2019-04-17 1.25 Crystal structure of NDM-1 complexed with compound 2
6D1C 2019-04-17 1.25 Crystal structure of NDM-1 complexed with compound 3
6D1A 2019-04-17 1.25 Crystal structure of NDM-1 complexed with compound 1
6O3R 2019-09-25 1.3 Crystal Structure of NDM-1 D199N with Compound 7
4RAW 2014-11-26 1.3 Crystal Structure of New Delhi Metallo-beta-Lactamase-1 Mutant M67V Complexed with Hydrolyzed Ampicillin
5O2E 2018-12-26 1.3 Crystal structure of NDM-1 in complex with hydrolyzed cefuroxime - new refinement
4RL0 2014-11-19 1.3 Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins
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
5WIG 2017-10-18 1.4 Structure of New Delhi Metallo-Beta-lactamase 4 (NDM-4)
6IBV 2019-05-15 1.4 Crystal structure of NDM-1 beta-lactamase in complex with broad spectrum boronic inhibitor cpd 1
6NY7 2019-09-25 1.4 Crystal Structure of NDM-1 D199N with Compound 16
6D1D 2019-04-17 1.4 Crystal structure of NDM-1 complexed with compound 6
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
4H0D 2012-09-26 1.5 New Delhi Metallo-beta-Lactamase-1 Complexed with Mn from Klebsiella pneumoniae
4HL1 2012-12-12 1.5 Crystal Structure of New Delhi Metallo-beta-Lactamase-1, Complexed with Cd and Ampicillin
5ZGQ 2018-08-22 1.5 Crystal structure of NDM-1 at pH7.5 (Tris-HCl, (NH4)2SO4) in complex with hydrolyzed ampicillin
4RAM 2014-09-24 1.5 Crystal Structure of New Delhi Metallo-beta-Lactamase-1 Mutant M67V Complexed with Hydrolyzed Penicillin G
6Q30 2019-04-24 1.5 Crystal structure of NDM-1 beta-lactamase in complex with boronic inhibitor cpd 5
6RMF 2019-09-11 1.51 Crystal structure of NDM-1 with VNRX-5133
5XP9 2018-03-07 1.55 Crystal structure of Bismuth bound NDM-1
5ZGU 2018-08-22 1.55 Crystal structure of NDM-1 at pH7.0 (HEPES) with 2 molecules per asymmetric unit
6MGY 2018-10-03 1.6 Crystal Structure of the New Deli Metallo Beta Lactamase Variant 5 from Klebsiella pneumoniae
6EFJ 2019-04-17 1.65 Crystal structure of NDM-1 with compound 9
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
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
4GYU 2012-09-26 1.8 Crystal Structure of New Delhi Metallo-beta-Lactamase-1 A121F mutant from Klebsiella pneumoniae
4EYF 2012-08-08 1.8 Crystal structure of NDM-1 bound to hydrolyzed benzylpenicillin
5N0H 2017-04-05 1.9 Crystal structure of NDM-1 in complex with hydrolyzed meropenem - new refinement
4EYL 2012-08-08 1.9 Crystal structure of NDM-1 bound to hydrolyzed meropenem
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
3RKJ 2011-05-18 2.0 Crystal Structure of New Delhi Metallo-Beta-Lactamase-1 from Klebsiella pnueumoniae
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)
4RL2 2014-11-19 2.01 Structural and mechanistic insights into NDM-1 catalyzed hydrolysis of cephalosporins
5O2F 2018-12-26 2.01 Crystal structure of NDM-1 in complex with hydrolyzed ampicillin - new refinement
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
6JKB 2020-03-25 2.44 Crystal structure of metallo-beta-lactamse, NDM-1, in complex with hydrolyzed ampicillin
3SRX 2011-07-20 2.5 New Delhi Metallo-beta-Lactamase-1 Complexed with Cd
5NBK 2018-10-03 2.6 NDM-1 metallo-beta-lactamase: a parsimonious interpretation of the diffraction data
5A5Z 2015-07-08 2.6 Approved Drugs Containing Thiols as Inhibitors of Metallo-beta- lactamases: Strategy To Combat Multidrug-Resistant Bacteria

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
200.0 A,B Metallo-beta-lactamase type 2 C7C422 BLAN1_KLEPN