Understanding the determinants of substrate specificity in IMP family metallo-β-lactamases: the importance of residue 262.


Abstract

In Gram-negative bacteria, resistance to β-lactam antibacterials is largely due to β-lactamases and is a growing public health threat. One of the most concerning β-lactamases to evolve in bacteria are the Class B enzymes, the metallo-β-lactamases (MBLs). To date, penams and cephems resistant to hydrolysis by MBLs have not yet been found. As a result of this broad substrate specificity, a better understanding of the role of catalytically important amino acids in MBLs is necessary to design novel β-lactams and inhibitors. Two MBLs, the wild type IMP-1 with serine at position 262, and an engineered variant with valine at the same position (IMP-1-S262V), were previously found to exhibit very different substrate spectra. These findings compelled us to investigate the impact of a threonine at position 262 (IMP-1-S262T) on the substrate spectrum. Here, we explore MBL sequence-structure-activity relationships by predicting and experimentally validating the effect of the S262T substitution in IMP-1. Using site-directed mutagenesis, threonine was introduced at position 262, and the IMP-1-S262T enzyme, as well as the other two enzymes IMP-1 and IMP-1-S262V, were purified and kinetic constants were determined against a range of β-lactam antibacterials. Catalytic efficiencies (kcat /KM ) obtained with IMP-1-S262T and minimum inhibitory concentrations (MICs) observed with bacterial cells expressing the protein were intermediate or comparable to the corresponding values with IMP-1 and IMP-1-S262V, validating the role of this residue in catalysis. Our results reveal the important role of IMP residue 262 in β-lactam turnover and support this approach to predict activities of certain novel MBL variants.

Submission Details

ID: qvtjSS8H

Submitter: Peter Oelschlaeger

Submission Date: Aug. 22, 2019, 12:36 p.m.

Version: 1

Publication Details
Pegg KM;Liu EM;George AC;LaCuran AE;Bethel CR;Bonomo RA;Oelschlaeger P,Protein Sci (2014) Understanding the determinants of substrate specificity in IMP family metallo-β-lactamases: the importance of residue 262. PMID:25131397
Additional Information

According to the standard numbering scheme (PMID 15215079) residue 196 is residue 262. ND indicates not detectable. S, I, and R indicate susceptible, intermediate, and resistant, respectively.

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
6JED 2019-08-07 1.57 Crystal structure of IMP-1 metallo-beta-lactamase in a complex with MCR
6LBL 2020-02-19 1.68 Crystal structure of IMP-1 metallo-beta-lactamase in complex with NO9 inhibitor
5Y5B 2018-08-08 1.7 Crystal Structure Of IMP-1 Metallo-beta-lactamase
4C1G 2014-08-27 1.71 Crystal structure of the metallo-beta-lactamase IMP-1 with D-captopril
5EV6 2016-06-01 1.98 Crystal structure of the native, di-zinc metallo-beta-lactamase IMP-1
1DD6 2000-11-08 2.0 IMP-1 METALLO BETA-LACTAMASE FROM PSEUDOMONAS AERUGINOSA IN COMPLEX WITH A MERCAPTOCARBOXYLATE INHIBITOR
5HH4 2017-01-18 2.0 Crystal structure of metallo-beta-lactamase IMP-1 in complex with a phosphonate-based inhibitor
1WUO 2005-03-29 2.01 Crystal structure of metallo-beta-lactamase IMP-1 mutant (D81A)
6JKA 2020-03-25 2.01 Crystal structure of metallo-beta-lactamse, IMP-1, in complex with a thiazole-bearing inhibitor
4C1F 2014-08-27 2.01 Crystal structure of the metallo-beta-lactamase IMP-1 with L-captopril
5EV8 2016-06-01 2.3 Crystal structure of the metallo-beta-lactamase IMP-1 in complex with the bisthiazolidine inhibitor D-CS319
5EWA 2016-06-01 2.3 Crystal structure of the metallo-beta-lactamase IMP-1 in complex with the bisthiazolidine inhibitor L-VC26
2DOO 2006-11-28 2.43 The structure of IMP-1 complexed with the detecting reagent (DansylC4SH) by a fluorescent probe
1VGN 2005-06-21 2.63 Structure-based design of the irreversible inhibitors to metallo--lactamase (IMP-1)
1WUP 2005-03-29 3.0 Crystal structure of metallo-beta-lactamase IMP-1 mutant (D81E)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 A Metallo-beta-lactamase type 2 P52699 BLAB_SERMA