Asiaticoside Attenuates Blood-Spinal Cord Barrier Disruption by Inhibiting Endoplasmic Reticulum Stress in Pericytes After Spinal Cord Injury.

The blood-spinal cord barrier (BSCB) plays a vital role in the recovery of spinal cord function after spinal cord injury (SCI). Pericytes, pluripotent members of the neurovascular unit (NVU), receive signals from neighboring cells and are critical for maintaining CNS function. Therapeutic targets for the BSCB include endothelial cells (ECs) and glial cells, but few drugs target pericytes. This study was designed to explore whether asiaticoside has a positively effect on pericytes and the integrity of the BSCB. In this study, we found that asiaticoside could inhibit the loss of junction proteins just 1 day after SCI in vivo, but our in vitro study showed no significant differences in the expression of endothelial junction proteins between the control and asiaticoside treatment groups. We also found that asiaticoside could inhibit endoplasmic reticulum (ER) stress and pericyte apoptosis, which might be associated with the inhibition of junction protein reduction in ECs. Thus, we investigated the interactions between pericytes and ECs. Our results showed that asiaticoside could decrease the release of matrix metalloproteinase (MMP)-9 in pericytes and therefore upregulate the expression of junction proteins in ECs. Furthermore, the protective effect of asiaticoside on pericytes is related to the inhibition of ER stress via the MAPK signaling pathway. Taken together, our results demonstrate that asiaticoside treatment inhibits BSCB disruption and enhances functional recovery after SCI.

Structure and function of the membrane microdomains in osteoclasts.

The cell membrane structure is closely related to the occurrence and progression of many metabolic bone diseases observed in the clinic and is an important target to the development of therapeutic strategies for these diseases. Strong experimental evidence supports the existence of membrane microdomains in osteoclasts (OCs). However, the potential membrane microdomains and the crucial mechanisms underlying their roles in OCs have not been fully characterized. Membrane microdomain components, such as scaffolding proteins and the actin cytoskeleton, as well as the roles of individual membrane proteins, need to be elucidated. In this review, we discuss the compositions and critical functions of membrane microdomains that determine the biological behavior of OCs through the three main stages of the OC life cycle.

Pregnane X receptor (PXR) deficiency protects against spinal cord injury by activating NRF2/HO-1 pathway.

INTRODUCTION: As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a ligand-activated nuclear receptor with a major regulatory role in xenobiotic and endobiotic metabolism and recently has been implicated in the central nervous system. In the present study, we aimed to investigate the role and mechanism of PXR in SCI. METHODS: The clip-compressive SCI model was performed in male wild-type C57BL/6 (PXR+/+ ) and PXR-knockout (PXR-/- ) mice. The N2a H2 O2 -induced injury model mimicked the pathological process of SCI in vitro. Pregnenolone 16α-carbonitrile (PCN), a mouse-specific PXR agonist, was used to activate PXR in vivo and in vitro. The siRNA was applied to knock down the PXR expression in vitro. Transcriptome sequencing analysis was performed to discover the relevant mechanism, and the NRF2 inhibitor ML385 was used to validate the involvement of PXR in influencing the NRF2/HO-1 pathway in the SCI process. RESULTS: The expression of PXR decreased after SCI and reached a minimum on the third day. In vivo, PXR knockout significantly improved the motor function of mice after SCI, meanwhile, inhibited apoptosis, inflammation, and oxidative stress induced by SCI. On the contrary, activation of PXR by PCN negatively influenced the recovery of SCI. Mechanistically, transcriptome sequencing analysis revealed that PXR activation downregulated the mRNA level of heme oxygenase-1 (HO-1) after SCI. We further verified that PXR deficiency activated the NRF2/HO-1 pathway and PXR activation inhibited this pathway in vitro. CONCLUSION: PXR is involved in the recovery of motor function after SCI by regulating NRF2/HO-1 pathway.

Taxifolin attenuates neuroinflammation and microglial pyroptosis via the PI3K/Akt signaling pathway after spinal cord injury.

Spinal cord injury (SCI) is a severe injury characterized by neuroinflammation and oxidative stress. Taxifolin is exhibits anti-inflammatory and antioxidative activities in neurologic diseases. However, the roles and mechanisms of taxifolin in neuroinflammation and microglial pyroptosis after SCI remain unclear. The present study aims to investigate the effect of taxifolin on SCI and its potential underlying mechanisms in in vivo and in vitro models. In this study, taxifolin markedly reduced microglial activation mediated oxidative stress, and inhibited the expression of pyroptosis-related proteins (NLRP3, GSDMD, ASC, and Caspase-1) and inflammatory cytokines (IL-1ß and IL-18) after SCI, as shown by immunofluorescence staining and western blot assays. In addition, taxifolin promoted axonal regeneration and improved functional recovery after SCI. In vitro studies showed that taxifolin attenuated the activation of microglia and oxidative stress after lipopolysaccharide (LPS) + adenosine-triphosphate (ATP) stimulation in BV2 cells. We also observed that taxifolin inhibited the pyroptosis-related proteins and reduced the release of inflammatory cytokines. Moreover, to explore how taxifolin exerts its effects on microglial pyroptosis and axonal regeneration of neurons, we performed an in vitro study in BV-2 cells and PC12 cells co-culture. The results revealed that taxifolin facilitated axonal regeneration of PC12 cells in co-culture with LPS + ATP-induced BV-2 cells. Mechanistically, taxifolin regulated microglial pyroptosis via the PI3K/AKT signaling pathway. Taken together, these results suggest that taxifolin alleviates neuroinflammation and microglial pyroptosis through the PI3K/AKT signaling pathway after SCI, and promotes axonal regeneration and improves functional recovery, suggesting that taxifolin may represent a potential therapeutic agent for SCI.

Efficacy of Ultrasound-Guided Quadratus Lumborum Block for Postoperative Analgesia After Hip Arthroplasty: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Ultrasound-guided quadratus lumborum (QL) block as a novel regional anesthetic technique was proposed in 2007 that can be applied in patients following hip arthroplasty. This study aimed to evaluate the efficacy of the QL block for pain control in patients undergoing hip arthroplasty. METHODS: We performed a comprehensive search of PubMed, Web of Science, Scopus, Cochrane Library, Embase databases, Google Scholar, and CNKI for randomized controlled trials up to December 2021. According to the inclusion and exclusion criteria established in advance, "QL block" and "hip arthroplasty" related MeSH terms and free-text words were used. RESULTS: Our meta-analysis included 11 randomized controlled trials involving a total of 830 patients between 2018 and 2021. The results indicated that compared to the non-QL block group, Visual Analog Scale (VAS) score at mobilization in the QL block group demonstrated statistical and clinical significance at all time points (12, 24, and 48 hours), but VAS score at rest failed to reach the MCID (minimal clinically important difference). Meanwhile, opioid consumption in the QL block group only demonstrated statistical and clinical significance at 48 hours postoperatively, but did not reach the MCID at 12 or 24 hours postoperatively. The QL block increased satisfaction scores. There was a statistically significant reduction in the incidence of postoperative nausea and vomiting, but no difference in the incidence of pruritus and urinary retention. CONCLUSION: The QL block significantly reduced postoperative VAS score at mobilization, and opioid consumption at 48 hours in patients after hip arthroplasty compared to no block, which reached the MCID. The QL block also decreased postoperative nausea and vomiting and increased satisfaction scores. Although these are promising results, the clinical relevance of the efficacy of the QL block remains to be further understood as larger studies are needed.

Volume-controlled ventilation versus pressure-controlled ventilation during spine surgery in the prone position: A meta-analysis.

Background: Many studies have investigated a comparison of the potency and safety of PCV versus VCV modes in spinal surgery in prone position. However, controversy about the maximal benefits of which ventilation modes remains. The main purpose of this meta-analysis was to investigate which one is the optimal ventilation for surgery patients undergoing spine surgery in prone position between the two ventilation modes as PCV and VCV. Methods: We conducted a comprehensive search of PubMed, Embase, Web of Science, the Cochrane Library, and Google Scholar for potentially eligible articles. The continuous outcomes were analyzed using the mean difference and the associated 95% confidence interval. Meta-analysis was performed using Review Manager 5.4 software. Results: Our meta-analysis included 8 RCTs involving a total of 454 patients between 2012 and 2020. The results demonstrated that IOB, Ppeak and CVP for VCV are significantly superior to PCV in spinal surgery in prone position. And PCV had higher Cdyn and PaO2/FiO2 than VCV. But there was no significant difference between PCV and VCV in terms of POB, Hb, HCT, HR and MAP. Conclusions: The PCV mode displayed a more satisfying effect than VCV mode. Compared to VCV mode in same preset of tidal volume, the patients with PCV mode in prone position demonstrated less IOB, lower Ppeak and CVP, and higher PaO2/FiO2 in spinal surgery. However, there is no obvious difference between PCV and VCV in terms of hemodynamics variables (HR and MAP).

Hydroxamate and thiosemicarbazone: Two highly promising scaffolds for the development of SARS-CoV-2 antivirals.

The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (Mpro) of SARS-CoV-2 is promising target for antiviral drugs, which plays a vital role for viral duplication. Development of the inhibitor against Mpro is an ideal strategy to combat COVID-19. In this work, twenty-three hydroxamates 1a-i and thiosemicarbazones 2a-n were identified by FRET screening to be the potent inhibitors of Mpro, which exhibited more than 94% (except 1c) and more than 69% inhibition, and an IC50 value in the range of 0.12-31.51 and 2.43-34.22 µM, respectively. 1a and 2b were found to be the most effective inhibitors in the hydroxamates and thiosemicarbazones, with an IC50 of 0.12 and 2.43 µM, respectively. Enzyme kinetics, jump dilution and thermal shift assays revealed that 2b is a competitive inhibitor of Mpro, while 1a is a time-dependently inhibitor; 2b reversibly but 1a irreversibly bound to the target; the binding of 2b increased but 1a decreased stability of the target, and DTT assays indicate that 1a is the promiscuous cysteine protease inhibitor. Cytotoxicity assays showed that 1a has low, but 2b has certain cytotoxicity on the mouse fibroblast cells (L929). Docking studies revealed that the benzyloxycarbonyl carbon of 1a formed thioester with Cys145, while the phenolic hydroxyl oxygen of 2b formed H-bonds with Cys145 and Asn142. This work provided two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.

Quinolinyl sulfonamides and sulphonyl esters exhibit inhibitory efficacy against New Delhi metallo-ß-lactamase-1 (NDM-1).

The "superbug" infection caused by metallo-ß-lactamases (MßLs) has grown into anemergent health threat, and development of effective MßL inhibitors to restore existing antibiotic efficacy is an ideal alternative. Although the serine-ß-lactamase inhibitors have been used in clinical settings, MßL inhibitors are not available to date. In this work, thirty-one quinolinyl sulfonamides 1a-p and sulphonyl esters 2a-o were synthesized and assayed against MßL NDM-1. The obtained molecules specifically inhibited NDM-1, 1a-p and 2a-o exhibited an IC50 value in the range of 0.02-1.4 and 8.3-24.8 µM, respectively, and 1e and 1f were found to be the most potent inhibitors, with an IC50 of 0.02 µM using meropenem (MER) as substrate. Structure-activity relationship reveals that the substitute phenyl and the phenyl with a halogen atom more significantly improve inhibitory effect of quinolinederivatives on NDM-1. 1a-p restored antimicrobial effect of MER on E. coli with NDM-1, EC01 and EC08, resulting in a 2-64-fold reduction in MIC values. Most importantly, 1e synergized MER and significantly reduced the load of EC08 in the spleen and liver of mice after a single intraperitoneal dose. Docking studies suggested that the endocyclic nitrogen of the quinoline ring, and exocyclic nitrogen of the sulfonamide functional group are coordinate with Zn(II) ion at active sites of NDM-1. Cytotoxicity assays indicated that 1e had low cytotoxicity. This work offers potential lead compounds for further development of the clinically useful inhibitor targeting NDM-1.

Efficacy of Thoracolumbar Interfascial Plane Block for Postoperative Analgesia in Lumbar Spine Surgery: A Meta-analysis of Randomized Clinical Trials.

BACKGROUND: Thoracolumbar interfascial plane (TLIP) block as a novel plane block technique was proposed in 2015 and can be performed in patients undergoing lumbar spine surgery. However, no meta-analysis demonstrates the effects of TLIP block on postoperative pain undergoing lumbar spine surgery. OBJECTIVES: The purpose of this study is to evaluate the postoperative analgesic efficacy of TLIP block with patient-controlled analgesia (PCA) undergoing lumbar spine surgery compared to be given PCA alone after lumbar spine surgery. STUDY DESIGN: This meta-analysis pooled all data published in randomized controlled trials (RCTs) examining the efficacy of TLIP following lumbar spine surgery. METHODS: We conducted a comprehensive search of PubMed, Web of Science, Embase databases, the Cochrane Library, and Google Scholar for randomized controlled trials (RCTs) up to December 2020. According to the inclusion and exclusion criteria established in advance, "TLIP" and "lumbar spine surgery" related MeSH terms and free-text words were used. All of the data on visual analog scales (VAS) scores, PCA compression frequency, PCA consumption, and nausea rates were reported. All analyses were performed with RevMan 5.4 software. RESULTS: A total of 9 RCTs with 618 patients meet the inclusion criteria. The results demonstrated that VAS scores for pain during movement and while at rest were markedly lower in the TLIP group than those in the control group in all the postoperative periods (1-2 h, 12 h, 18 h, and 24 h) (P < 0.05). VAS scores at rest 1-2 h postoperatively (MD: -2.16; 95% CI: [-3.86, -0.46]); 12 h (MD: -1.22; 95% CI: [-2.33, -0.11]); 18 h (MD: -1.40; 95% CI: [-1.55, -1.24]); 24 h (MD: -1.38; 95% CI: [-1.94, -0.81]); VAS scores at movement 1-2 postoperatively (MD: -2.26; 95% CI: [-4.28, -0.23]); 12 h (MD: -2.11; 95% CI: [-3.13, -1.10]); 18 h (MD: -1.63; 95% CI: [-1.77, -1.48]); 24 h (MD: -1.47; 95% CI: [-1.98, -0.95]). Meanwhile, PCA compression frequency, PCA consumption, and nausea rates were significantly lower in the TLIP group after lumbar spine surgery (P < 0.05): PCA compressions frequency (MD: -4.08; 95% CI: [-5.28, -2.88]); PCA consumption (MD: -14.30; 95% CI: [-20.68, -7.92]); nausea rates (RR: 0.47; 95% CI: [0.32, 0.68]). LIMITATIONS: Despite 9 RCTs, the sample size was still small, so more high-quality RCTs with large samples will be urgently required for stronger evidence to support TLIP block in lumbar spine surgery. CONCLUSIONS: The TLIP block is an effective strategy to improve postoperative pain at rest/movement and to reduce PCA consumption in patients undergoing lumbar spine surgery, which exerts significant analgesia. In the future, it is worth being applied in lumbar spine surgery extensively.

Hippocampal Aromatase Knockdown Aggravates Ovariectomy-Induced Spatial Memory Impairment, Aß Accumulation and Neural Plasticity Deficiency in Adult Female Mice.

Ovarian estrogens (mainly 17ß estradiol, E2) have been involved in the regulation of the structure of hippocampus, the center of spatial memory. In recent years, high levels of aromatase (AROM), the estrogen synthase, has been localized in hippocampus; and this hippocampus-derived E2 seems to be functional in synaptic plasticity and spatial memory as ovarian E2 does. However, the contribution of ovarian E2 and hippocampal E2 to spatial memory and neural plasticity remains unclear. In this study, AROM-specific RNA interference AAVs (shAROM) were constructed and injected into the hippocampus of control or ovariectomized (OVX) mice. Four weeks later the spatial learning and memory behavior was examined with Morris water maze, the expression of hippocampal Aß related proteins, selected synaptic proteins and CA1 synapse density, actin polymerization related proteins and CA1 spine density were also examined. The results showed that while OVX and hippocampal shAROM contributed similarly to most of the parameters examined, shAROM induced more increase in BACE1 (amyloidogenic ß-secretase), more decrease in neprilysin (Aß remover) and Profilin-1 (actin polymerization inducer). More importantly, combined OVX and shAROM treatment displayed most significant impairment of spatial learning and memory as well as decrease in synaptic plasticity compared to OVX or shAROM alone. In conclusion, the above results clearly demonstrated the crucial role of hippocampal E2 in the regulation of the structure and function of hippocampus besides ovarian E2, indicating that hippocampal E2 content should also be taken into consideration during estrogenic replacement.

Exploring the Activation Mechanism of a Metabotropic Glutamate Receptor Homodimer via Molecular Dynamics Simulation.

Metabotropic glutamate receptors of class C GPCRs exist as constitutive dimers, which play important roles in activating excitatory synapses of the central nervous system. However, the activation mechanism induced by agonists has not been clarified in experiments. To address the problem, we used microsecond all-atom molecular dynamics (MD) simulation couple with protein structure network (PSN) to explore the glutamate-induced activation for the mGluR1 homodimer. The results indicate that glutamate binding stabilizes not only the closure of Venus flytrap domains but also the polar interaction of LB2-LB2, in turn keeping the extracelluar domain in the active state. The activation of the extracelluar domain drives transmembrane domains (TMDs) of the two protomers closer and induces asymmetric activation for the TMD domains of the two protomers. One protomer with lower binding affinity to the agonist is activated, while the other protomer with higher binding energy is still in the inactive state. The PSN analysis identifies the allosteric regulation pathway from the ligand-binding pocket in the extracellular domain to the G-protein binding site in the intracellular TMD region and further reveals that the asymmetric activation is attributed to a combination of trans-pathway and cis-pathway regulations from two glumatates, rather than a single activation pathway. These observations could provide valuable molecular information for understanding of the structure and the implications in drug efficacy for the class C GPCR dimers.

Loureirin B Promotes Axon Regeneration by Inhibiting Endoplasmic Reticulum Stress: Induced Mitochondrial Dysfunction and Regulating the Akt/GSK-3ß Pathway after Spinal Cord Injury.

Axon retraction greatly limits functional recovery after spinal cord injury (SCI) and neuron polarization, which affects processes including axon formation and development, is a promising target for promoting axon regeneration. Increasing microtubule stability has been demonstrated to improve intrinsic axon regeneration processes and is critically related to endoplasmic reticulum (ER)-mitochondria interactions. We used real-time polymerase chain reaction, Western blotting, and immunofluorescence to screen a variety of natural compounds, and found that Loureirin B (LrB) effectively promoted neuron polarization and axon regeneration in vitro and in vivo. LrB significantly inhibited ER stress and thereby promoted mitochondrial functions by regulating mitochondrial fusion. Further, LrB reactivated the Akt/GSK-3ß pathway, which plays critical roles in cell survival and microtubule stabilization. Taken together, our results suggest that the effects of LrB on neuron regeneration involve the inhibition of ER stress-induced mitochondrial dysfunction and activation of the Akt/GSK-3ß pathway, which further promotes microtubule stabilization. LrB may therefore be a promising candidate for facilitating recovery following SCI.

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies.

The metallo-ß-lactamases (MßLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in ß-lactam-containing antibiotics, and render the antibiotics inactive. All known MßLs 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 MßL 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.

Stepwise assembly of the human replicative polymerase holoenzyme.

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.

The human lagging strand DNA polymerase δ holoenzyme is distributive.

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5'-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ(AA). The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.

Single-molecule studies of DNA replisome function.

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.

Motion of the zinc ions in catalysis by a dizinc metallo-beta-lactamase.

We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-beta-lactamase (MbetaL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MbetaL move away from each other, by approximately 0.3 A after 10 ms of reaction with nitrocefin, from 3.4 to 3.7 A. Together with our previous characterization of the resting enzyme and its nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 A, 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-beta-lactamase catalysis.

Differential binding of Co(II) and Zn(II) to metallo-beta-lactamase Bla2 from Bacillus anthracis.

In an effort to probe the structure, mechanism, and biochemical properties of metallo-beta-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, (1)H 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.

Structure and mechanism of copper- and nickel-substituted analogues of metallo-beta-lactamase L1.

In an effort to further probe metal binding to metallo-beta-lactamase L1 (mbetal 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 k(cat) (96 s(-1)) and K(m) (224 microM) 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 (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 mbetal 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 beta-lactam-containing antibiotics.

Role of the Zn1 and Zn2 sites in metallo-beta-lactamase L1.

In an effort to probe the role of the Zn(II) sites in metallo-beta-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.