In an effort to probe the role of the Zn(II) sites in metallo-β-lactamase L1, mononuclear metal ion containing and heterobimetallic analogs of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn 1 site, was shown to be slightly active; however, this enzyme did not stabilize a nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)-and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn 2 site in L1. Heterobimetallic analogs (ZnCo and ZnFe) analogs of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogs were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) 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. These studies demonstrate that the metal ion in the Zn 1 site is essential for catalysis in L1 and that the metal ion in the Zn 2 site is crucial for stabilization of the nitrocefinderived reaction intermediate.
In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication.DOI: http://dx.doi.org/10.7554/eLife.00278.001
In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was over-expressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 eq of Zn(II). Steady-state kinetic studies showed that mono-Zn(II) Bla2 (1Zn-Bla2) is active, while di-Zn(II) Bla2 (ZnZn-Bla2) was unstable. Catalytically, 1Zn-Bla2 behaves like the related enzymes CcrA and L1. In contrast, diCo(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analog. Rapid kinetics and UV-Vis, 1 H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distrubuted, while EXAFS shows that Zn(II) binding is sequential. To our knowledge, this is the first documented example of a Zn enzyme that binds Co(II) and Zn(II) via distinct mechanisms, underscoring the need to demonstrate transferability when extrapolating results on Co(II)-substituted proteins to the native Zn(II)-containing forms.
In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was over-expressed as mature protein (M-L1) and full-length (FL-L1) analogs, and the analogs were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of E. coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was over-expressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analog was obtained. On the other hand, M-L1, lacking the putative leader sequence, was localized in the cytoplasm of E. coli and shown to bind various amounts of Fe and Zn(II), and like FL-L1, the metal content of the resulting enzyme could be affected by the amount of metal ions in the growth medium. L1 was refolded in the presence of Fe, and a dinuclear Fe-containing analog of L1 was obtained, although this analog is catalytically-inactive. EPR spectra demonstrate the presence of an antiferromagnetically-coupled Fe(III)Fe(II) center in Fe-containing L1 and suggests the presence of a Fe(III)Zn(II) center in M-L1. Metal analyses on the cytoplasmic and periplasmic fractions of E. coli showed that the concentration of metal ions in the periplasm is not tightly controlled and increases as the concentration of metal ions in the growth medium increases. In contrast, the concentration of Zn(II) in the cytoplasm is tightly-controlled while that of Fe is less so.Bacterial resistance to β-lactam containing antibiotics such as penicillins, cephalosporins, and carbapenems is most often accomplished by expression of β-lactamases, which hydrolyze the C-N bond of these antibiotics ( 1-4 ). A majority of these β-lactamases utilize an active site serine group for the nucleophilic attack on the β-lactam carbonyl, and the serine β-lactamases have been studied extensively for many years ( 4 ). On the other hand, one class (Class B) of β-lactamases utilizes a metal-assisted hydrolysis pathway to inactivate β-lactam containing antibiotics, and these enzymes are called metallo-β-lactamases (mβl's) ( 1, 2, 5-7 ). The mβl's have been further divided into subgroups based on sequence identity, Zn(II) content, substrate preference, and biochemical properties. Subgroup B1 enzymes require 2 Zn(II) ions for full catalytic activity, exhibit kinetic preference for penicillins as substrates, exhibit >23% sequence identity toward other subgroup B1 members, and are represented by mβl's CcrA from Bacteroides fragilis, BcII from Bacillus cereus, and IMP-1 from various sources ( 1, 5 ). Subgroup B2 enzymes require only 1 Zn(II) ion for full catalytic activity, preferentially hydrolyze carbapenems, exhibit 11% sequence identity with the subgroup B1 enzymes, and
We report rapid-freeze-quench X-ray absorption spectroscopy of a di-zinc metallo-β-lactamase (MβL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MβL move away from each other, by ~ 0.3 Å after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 Å. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 Å, these data indicate a scissoring motion of the active site that accompanies the ring-opening step. The average Zn(II) coordination number of 4.5 in the resting enzyme appears to be maintained throughout the reaction with nitrocefin. This is the first direct structural information available on early stage di-zinc metallo-β-lactamase catalysis.
Metallo-β-lactamases are responsible for conferring antibiotic resistance on certain pathogenic bacteria. In consequence, the search for inhibitors that may be useful in combating antibiotic resistance has fueled much study of the active sites of these enzymes. There exists circumstantial evidence that the binding of substrates and inhibitors to metallo-β-lactamases may involve binding to the organic part of the molecule, in addition to or prior to binding to one or more active site metal ions. It has also been postulated that a conformational change may accompany this putative binding. In the present study, electron paramagnetic resonance spectrokinetic study of a spin-labeled variant of the class B2 metallo-β-lactamase ImiS identified movement of a component residue on a conserved α-helix in a catalytically competent time upon formation of a transient reaction intermediate with the substrate imipenem. In a significant subpopulation of ImiS, this conformational change was not associated with substrate binding to the active site metal ion but, rather, represents a distinct step in the reaction with ImiS. This observation has implications regarding the determinants of substrate specificity in metallo-β-lactamases and the design of potentially clinically useful inhibitors.
In an effort to further probe metal binding to metallo-β-lactamase L1 (mβl L1), Cu-(Cu-L1) and Nisubstituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equivalents of Cu and small amounts of Zn(II) and Fe. The EPR spectrum of Cu-L1 exhibited two overlapping, axial signals, indicative of type 2 sites with distinct affinities for Cu(II). Both signals indicated multiple nitrogen ligands. Despite the expected proximity of the Cu(II) ions, however, only indirect evidence was found for spin-spin coupling. Cu-L1 exhibited higher k cat (96 s −1 ) and K m (224 μM) values, as compared to the values of dinuclear Zn(II)-containing L1, when nitrocefin was used as substrate. The Ni-L1 bound 1 equivalent of Ni and 0.3 equivalents of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by 1 H NMR spectra, which showed relatively sharp proton resonances. Stopped-flow kinetic studies showed that ZnNi-L1 stabilized significant amounts of the nitrocefin-derived intermediate and that the decay of intermediate is rate-limiting. 1 H NMR spectra demonstrate that Ni(II) binds in the Zn 2 site and that the ring-opened product coordinates Ni(II). Both Cu-L1 and ZnNi-L1 hydrolyze cephalosporins and carbapenems, but not penicillins, suggesting that the Zn 2 site modulates substrate preference in mβ1 L1. These studies demonstrate that the Zn 2 site in L1 is very flexible and can accommodate a number of different transition metal ions; this flexibility could possibly offer an organism that produces L1 an evolutionary advantage when challenged with β-lactam containing antibiotics.β-Lactams are inexpensive and widely-used antibiotics against microbes since the 1940's(1). There are three different major classes of β-lactams, penicillins, cephalosporins, and carbapenems, that have been used clinically. However, most microorganisms have obtained the ability to either pump the β-lactams out of the cell via transporter proteins (2) or to hydrolyze these compounds by secreting β-lactamases into the periplasm or milieu (3). Four distinct classes of β-lactamases have been identified (4). Unlike class A, C, and D β-lactamases, which utilize an active site serine as a nucleophile, class B β-lactamases, metallo-β-lactamases or Mβl's, are a group of enzymes that require Zn(II) to hydrolyze β-lactams (5). There have been >50 Mβl's identified and categorized into three subgroups, according to amino acid sequence homology, the requirement of Zn(II) (1 or 2) for maximal activity, the identity of the metal
In an effort to overcome previous problems with the preparation of Co(II)-substituted metallo-beta-lactamase L1, two strategies were undertaken. Attempts to prepare Co(II)-substituted L1 using biological incorporation resulted in an enzyme that contained only 1 Eq of cobalt and exhibited no catalytic activity. Co(II)-substituted L1 could be prepared by refolding metal-free L1 in the presence of Co(II), and the resulting enzyme contained 1.8 Eq of cobalt, yielded a UV-Vis spectrum consistent with 5-coordinate Co(II), and exhibited a k(cat) of 63 s(-1) and K(m) of 20 microM when using nitrocefin as the substrate. Pre-steady-state fluorescence and UV-Vis studies demonstrated that refolded, Co(II)-substituted L1 uses the same kinetic mechanism as Zn(II)-containing L1, in which a reaction intermediate is formed when using nitrocefin as substrate. The described refolding strategy can be used to prepare other Co(II)-substituted Zn(II)-metalloenzymes, particularly those that contain a solvent-exposable disulfide, which often causes oxidation of Co(II) to Co(III).
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