Role of the Zn\u3csub\u3e1\u3c/sub\u3e and Zn\u3csub\u3e2\u3c/sub\u3e Sites in Metallo-β-lactamase L1

In an effort to probe the role of the Zn(II) sites in metallo-β-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 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 Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues 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 analogues were reacted with nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme−product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the nitrocefin-derived reaction intermediate

Conformational Changes in the Metallo-β-lactamase ImiS During the Catalytic Reaction: An EPR Spectrokinetic Study of Co(II)-Spin Label Interactions

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

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

We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc 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 dizinc metallo-β-lactamase catalysis

Metal Content of Metallo-β-lactamase L1 Is Determined by the Bioavailability of Metal Ions

In an effort to probe whether the metal content of metallo-β-lactamase L1 is affected by metal ion bioavailability, L1 was overexpressed as mature protein (M-L1) and full-length (FL-L1) analogues, and the analogues were characterized with metal analyses, kinetics, and EPR spectroscopy. FL-L1, containing the putative leader sequence, was localized in the periplasm of Escherichia coli and shown to bind Zn(II) preferentially. The metal content of FL-L1 could be altered if the enzyme was overexpressed in minimal medium containing Fe and Mn, and surprisingly, an Fe-binding analogue 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 analogue of L1 was obtained, although this analogue is catalytically inactive. EPR spectra demonstrate the presence of an antiferromagnetically coupled Fe(III)Fe(II) center in Fe-containing L1 and suggest 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

Structure and Mechanism of Copper- and Nickel-Substituted Analogues of Metallo-β-lactamase L1

In an effort to further probe metal binding to metallo-β-lactamase L1 (mβl L1), Cu- (Cu-L1) and Ni-substituted (Ni-L1) L1 were prepared and characterized by kinetic and spectroscopic studies. Cu-L1 bound 1.7 equiv 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 kcat (96 s−1) and Km (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 equiv of Ni and 0.3 equiv of Zn(II). Ni-L1 was EPR-silent, suggesting that the oxidation state of nickel was +2; this suggestion was confirmed by 1H 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. 1H NMR spectra demonstrate that Ni(II) binds in the Zn2 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 Zn2 site modulates substrate preference in mβl L1. These studies demonstrate that the Zn2 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

Differential Binding of Co(II) and Zn(II) to Metallo-β-Lactamase Bla2 from \u3cem\u3eBacillus anthracis\u3c/em\u3e

In an effort to probe the structure, mechanism, and biochemical properties of metallo-β-lactamase Bla2 from Bacillus anthracis, the enzyme was overexpressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equiv 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, di-Co(II) Bla2 (CoCo-Bla2) is substantially more active than the mono-Co(II) analogue. Rapid kinetics and UV−vis, 1H NMR, EPR, and EXAFS spectroscopic studies show that Co(II) binding to Bla2 is distributed, 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

Hot subdwarf stars identified in LAMOST DR8 with single-lined and composite spectra

222 hot subdwarf stars were identified with LAMOST DR8 spectra, among which 131 stars show composite spectra and have been decomposed, while 91 stars present single-lined spectra. Atmospheric parameters of all sample stars were obtained by fitting Hydrogen (H) and Helium (He) line profiles with synthetic spectra. Two long-period composite sdB binaries were newly discovered by combining our sample with the non-single star data from Gaia DR3. One of the new systems presents the highest eccentricity (i.e., 0.5 +/- 0.09) among known wide sdB binaries, which is beyond model predictions. 15 composite sdB stars fall in the high probability binary region of RUWE-AEN plane, and deserve priority follow-up observations to further study their binary nature. A distinct gap is clearly presented among temperatures of cool companions for our composite-spectra sample. But we could not come to a conclusion whether this feature is connected to the formation history of hot subdwarf stars before their binary natures are confirmed.Comment: 21 pages, 11 figures, 3 tables, Accepted for publication in Ap

Dilution of Dipolar Interactions in a Spin-labeled, Multimeric Metalloenzyme for DEER Studies

The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at positions 163 and 286 and analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling

Straw and phosphorus applications promote maize (Zea mays L.) growth in saline soil through changing soil carbon and phosphorus fractions

IntroductionStraw return has been widely recognized as an important carbon (C) enhancement measure in agroecosystems, but the C-phosphorus (P) interactions and their effects on plants in saline soils are still unclear.MethodsIn this study, we investigated the effects of straw return and three P application levels, no P fertilizer (Non-P), a conventional application rate of P fertilizer (CP), and a high application rate of P fertilizer (HP), on maize growth and soil C and P fractions through a pot experiment.Results and discussionThe results revealed that the dry matter weight of maize plant was no difference between the two straw return levels and was 15.36% higher under HP treatments than under Non-P treatments. Plant nutrient accumulations were enhanced by straw addition and increased with increasing P application rate. Straw application reduced the activities of peroxidase (POD), superoxide dismutase (SOD), catalase, and the content of malondialdehyde (MDA) in maize plants by 31.69%, 38.99%, 45.96% and 27.04%, respectively. P application decreased SOD, POD activities and MDA content in the absence of straw. The contents of easily oxidized organic carbon (EOC), particulate organic carbon (POC) and the ratio of POC/SOC in straw-added soils were 10.23%, 17.00% and 7.27% higher, respectively, than those in straw-absent soils. Compared with Non-P treatments, HP treatments led to an increase of 12.05%, 23.04% in EOC, POC contents respectively, while a decrease of 18.12% in the contribution of MAOC to the SOC pool. Straw return improved the P status of the saline soil by increasing soil available P (14.80%), organic P (35.91%) and Ca2-P contents (4.68%). The structural equation model showed that straw and P applications could promote maize growth (indicated by dry matter weight, P accumulation, antioxidant enzyme activity and MDA content) through improving soil C and P availabilities.ConclusionThis study provides evidence that straw return together with adequate P supply in saline soil can promote crop nutrient accumulation, attenuate the oxidation damage on crop growth, and be beneficial for SOC turnover and soil P activation