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dc.contributor.authorSkagseth, Susann
dc.contributor.authorChristopeit, Tony
dc.contributor.authorAkhter, Sundus
dc.contributor.authorBayer, Annette
dc.contributor.authorSamuelsen, Ørjan
dc.contributor.authorLeiros, Hanna-Kirsti S.
dc.date.accessioned2018-06-29T12:51:48Z
dc.date.available2018-06-29T12:51:48Z
dc.date.issued2017-05-30
dc.description.abstractMetallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant (KD) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than L-captopril (IC50 = 47 μM; KD = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than L-captopril.en_US
dc.descriptionSource at <a href=https://doi.org/10.1128/AAC.02602-16> https://doi.org/10.1128/AAC.02602-16</a>.en_US
dc.identifier.citationSkagseth, S., Christopeit, T., Akhter, S., Bayer, A., Samuelsen, Ø. & Leiros, H. (2017). Structural insights into TMB-1 and the role of residues 119 and 228 in substrate and inhibitor binding. Antimicrobial Agents and Chemotherapy, 61, e02602, 16(8), 1-19. https://doi.org/10.1128/AAC.02602-16en_US
dc.identifier.cristinIDFRIDAID 1501069
dc.identifier.doi10.1128/AAC.02602-16
dc.identifier.issn0066-4804
dc.identifier.issn1098-6596
dc.identifier.urihttps://hdl.handle.net/10037/13063
dc.language.isoengen_US
dc.publisherAmerican Society for Microbiologyen_US
dc.relation.journalAntimicrobial Agents and Chemotherapy
dc.relation.projectIDinfo:eu-repo/grantAgreement/RCN/FRIMEDBIO/213808/Norway/Combating Antibiotic Resistance - Development of Inhibitors against Antibiotic Resistance Enzymes//en_US
dc.relation.projectIDinfo:eu-repo/grantAgreement/RCN/SYNKNØYT/247732/Norway/Reisestøtte, synkrotron- og nøytronforskning, 2015-2017//en_US
dc.relation.projectIDinfo:eu-repo/grantAgreement/RCN/SYNKNØYT/218539/Norway/High Throughput Pipeline for Structure Based Drug Design, and Antibiotic Resistance Enzymes in particular//en_US
dc.rights.accessRightsopenAccessen_US
dc.subjectVDP::Matematikk og Naturvitenskap: 400::Kjemi: 440en_US
dc.subjectVDP::Mathematics and natural science: 400::Chemistry: 440en_US
dc.subjectmetallo-β-lactamaseen_US
dc.subjectTMB-1en_US
dc.subjectTMB-2en_US
dc.subjectthermal stabilityen_US
dc.subjectenzyme kineticsen_US
dc.subjectcrystal structureen_US
dc.subjectmutantsen_US
dc.titleStructural insights into TMB-1 and the role of residues 119 and 228 in substrate and inhibitor bindingen_US
dc.typeJournal articleen_US
dc.typeTidsskriftartikkelen_US
dc.typePeer revieweden_US


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