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Mechanistic Insights into ?-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation.

ABSTRACT: ?-Lactamases are a major threat to the clinical use of carbapenems, which are often antibiotics of last resort. Despite this, the reaction outcomes and mechanisms by which ?-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a ?-lactam ring fused to a pyrroline ring. Following ?-lactamase-mediated opening of the ?-lactam, the pyrroline may interconvert between an enamine (2-pyrroline) form and two epimeric imine (1-pyrroline) forms; previous crystallographic and spectroscopic studies have reported all three of these forms in the contexts of hydrolysis by different ?-lactamases. As we show by NMR spectroscopy, the serine ?-lactamases (KPC-2, SFC-1, CMY-10, OXA-23, and OXA-48) and metallo-?-lactamases (NDM-1, VIM-1, BcII, CphA, and L1) tested all degrade carbapenems to preferentially give the ?² (enamine) and/or (R)-?¹ (imine) products. Rapid non-enzymatic tautomerisation of the ?² product to the (R)-?¹ product prevents assignment of the nascent enzymatic product by NMR. The observed stereoselectivity implies that carbapenemases control the form of their pyrroline ring intermediate(s)/product(s), thereby preventing pyrroline tautomerisation from inhibiting catalysis.

SUBMITTER: Lohans CT

PROVIDER: S-EPMC6754457 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation.

Lohans Christopher T CT Freeman Emily I EI Groesen Emma van EV Tooke Catherine L CL Hinchliffe Philip P Spencer James J Brem Jürgen J Schofield Christopher J CJ

Scientific reports 20190920 1

β-Lactamases are a major threat to the clinical use of carbapenems, which are often antibiotics of last resort. Despite this, the reaction outcomes and mechanisms by which β-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a β-lactam ring fused to a pyrroline ring. Following β-lactamase-mediated opening of the β-lactam, the pyrroline may interconvert between an enamine (2-pyrroline) form and two epimeric imine (1-pyrroline) forms; previous c ...[more]

PMID: 31541180

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Project description:Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis-diol, is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDOKF1). TPDOKF1 exhibited substrate specificity for TPA (kcat/Km = 57 ± 9 mM-1 s-1). The TPDOKF1 structure harbors characteristic RO features as well as a unique catalytic domain that rationalizes the enzyme's function. The docking and mutagenesis studies reveal that its substrate specificity for TPA is mediated by the Arg309 and Arg390 residues, positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, as its mutation to alanine decreases the activity (kcat) by 80%. This study delineates the structural features that dictate the substrate recognition and specificity of TPDO. IMPORTANCE Global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential for tackling this. Microbial utilization of this released product, TPA, is an emerging and promising strategy for waste-to-value creation. Research in the last decade has identified terephthalate dioxygenase (TPDO) as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Dataset Information

Mechanistic Insights into ?-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation.

Publications

Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation.

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