Probing substrate binding to the metal binding sites in metallo-β-lactamase L1 during catalysis

Abstract

Metal ions in metallo-β-lactamases (MBLs) play a major role in catalysis. In this study we investigated the role of the metal ions in the Zn1 and Zn2 sites of MBL L1 during catalysis. A ZnCo (with Zn(II) in the invariant Zn1 site and Co(II) in the Zn2 site) analog of MBL L1 was prepared by using a biological incorporation method. Extended X-ray Absorption Fine Structure (EXAFS) spectroscopic studies were used to confirm that the ZnCo analog was prepared. To study the roles of the Zn(II) and Co(II) ions during catalysis, rapid freeze quench (RFQ)-EXAFS studies were used to probe the reaction of the ZnCo-L1 analog with chromacef when quenched at 10 ms, 50 ms, and 100 ms. The L1-product complex was also analyzed with EXAFS spectroscopy. The data show that the Zn-Co distance is 3.49 Å in the resting enzyme and that this distance increases by 0.3 Å in the sample that was quenched at 10 ms. The average Zn-Co distance decreases at the other time points until reaching a distance of 3.58 Å in the L1-product complex. The data also show that a Co-S interaction is present in the 100 ms quenched sample and in the L1-product complex, which suggests that there is a significant rearrangement of product in the active site.

Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

“…The pro -( R ) boron-bound exocyclic oxygen bridges Zn1 and Asp 120 , mimicking the proposed position of the hydroxide-derived oxygen in the oxyanion intermediate. Overall, the complexes of 2 with VIM-2 and BcII reveal that binding of the cyclic boronates closely mimics that proposed for the high-energy tetrahedral intermediate in MBL catalysis 21 ( Fig. 2a,e ), both in terms of structure and bonding.…”

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-β-Lactamases

“…Such changes in the positioning of metal ions induced by inhibitor–substrate interactions in β-lactamase catalysis have also been observed with extended X-ray absorption fine structure (EXFAS) spectroscopy studies, as shown in the case of the subclass B3 MBL L1 from S. maltophilia and with other metallo-enzymes (see e.g., − ); it may also be that the extent of such metal ion translocations is not fully reflected in crystallographic compared to solution studies. It should also be noted that the precipitants used in crystallography experiments for NDM-1: VNRX-5133 and OXA-10: VNRX-5133 structures were at pH 5.8 and pH 8.0, respectively.…”

Structure, function, and evolution of metallo-β-lactamases from the B3 subgroup—emerging targets to combat antibiotic resistance

“… a) B3 variants with the canonical HHH/DHH motif. The most studied representatives from the B3 MBL subgroup are L1 from S. maltophilia ( Simm et al, 2002 ; Aitha et al, 2016 ; Kim et al, 2020 ) and AIM-1 from P. aeruginosa ( Selleck et al, 2016 ), with the latter posing a major threat to healthcare due to its high catalytic efficiency towards a broad range of β-lactam antibiotics ( Supplementary Table S1 ). The metal ion in the Zn1 site is tetrahedrally coordinated by the three histidine ligands of the active site motif, as well as the oxygen from the hydroxide that in the resting enzyme bridges the two metal ions and which acts as the nucleophile that initiates the hydrolysis of the β-lactam bond.…”