Motion of the Zinc Ions in Catalysis by a Dizinc Metallo-β-Lactamase

Breece, Robert M.; Hu, Zhenxin; Bennett, Brian; Crowder, Michael W.; Tierney, David L.

doi:10.1021/ja902534b

Cited by 32 publications

(40 citation statements)

References 15 publications

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“…Inclusion of a metal-metal interaction at 3.81 Å to the model improved the fit residual by 34% (Figure S4 and Table S4). A similar increased metal-metal distance was previously observed in the ZnZn-L1/nitrocefin sample quenched at 10 ms. 18 Our stopped-flow kinetic studies suggest that the sample quenched at 10 ms contains predominantly enzyme-intermediate complex (Figure 2). These data strongly suggest that there was no change in the coordination number of either metal ion in the sample quenched at 10 ms.…”

Section: Resultsmentioning

confidence: 76%

“…20 In contrast, RFQ-EXAFS studies on ZnZn-L1 indicated an increase in the average coordination number of Zn(II) in the sample freeze quenched at 10 ms and in the product complex, as compared to the resting enzyme. 18 The EXAFS studies indicated an increase in the metal-metal distance in L1 after 10 ms of reaction with nitrocefin. The EXAFS studies indicated formation of a Zn-S interaction in the product complex, 18, 23 afforded by rotation around the C6-C7 bond after hydrolysis.…”

Section: Introductionmentioning

confidence: 96%

“…17–22 RFQ-EXAFS and RFQ-EPR studies showed different coordination numbers for the metal ions in L1 samples freeze quenched at 10 ms. 18, 20 RFQ-EPR studies on ZnCo-L1 demonstrated that Co(II) (in the Zn 2 site) is five-coordinate in the resting state, proceeds through a four-coordinate species during the reaction, and is five-coordinate in the enzyme product complex. 20 In contrast, RFQ-EXAFS studies on ZnZn-L1 indicated an increase in the average coordination number of Zn(II) in the sample freeze quenched at 10 ms and in the product complex, as compared to the resting enzyme.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Resultsmentioning

confidence: 76%

Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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“…50 This plasticity can be required for the different steps of the full catalytic cycle or it can also promote catalytic promiscuity. In particular, the mechanistic implication of the motion of the zinc ions dizinc metallo-beta-lactamases was already observed by Breece et al 51 Thus, keeping in mind that the structural experimental evidences are based on the apo enzyme or on a complex not corresponding to the reactants or intermediate, none of the obtained structures could be, a priori, discarded. Hamiltonian, was found that movement of Zn ions is dramatic from R to TS.…”

Section: Discussionmentioning

confidence: 99%

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase

Meliá

Ferrer

Moliner

et al. 2015

Archives of Biochemistry and Biophysics

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In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear β-lactamase, CphA from Aeromonas hydrophila. According to our results, significant different transition state structures are obtained for the hydrolysis of both antibiotics:while the TS of the CEF is a ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. This feature of the β-lactamase would be in turn, associated to the different mechanisms that the protein employs to hydrolyze the different antibiotics; i.e. the catalytic promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study will be used to shed some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MβL inhibitors against a broad spectrum of bacterial pathogens.

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“…[101] It was shown that loop movements occur during catalysis, and it has previously been shown that there are metal-metal distance changes between resting, intermediate-bound, and product-bound states; it seems possible that these two factors are linked. [102][103][104] It is thought that the flexibility in coordination states observed in MbL enables hydrolysis with different compounds, but at the cost of efficient hydrolysis. [100] The promiscuous reactions that different MbLs catalyse generally have lower k cat , where it is hypothesised that promiscuous substrates may not be well positioned relative to the nucleophilic hydroxide for catalysis.…”

Section: Active Site Changes and Activity Divergencementioning

confidence: 99%

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

et al. 2016

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An increasing number of bacterial metalloenzymes have been shown to catalyse the breakdown of xenobiotics in the environment, while others exhibit a variety of promiscuous xenobiotic-degrading activities. Several different evolutionary processes have allowed these enzymes to gain or enhance xenobiotic-degrading activity. In this review, we have surveyed the range of xenobiotic-degrading metalloenzymes, and discuss the molecular and catalytic basis for the development of new activities. We also highlight how our increased understanding of the natural evolution of xenobiotic-degrading metalloenzymes can be been applied to laboratory enzyme design.

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Motion of the Zinc Ions in Catalysis by a Dizinc Metallo-β-Lactamase

Cited by 32 publications

References 15 publications

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

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

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

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