Heterologous Expression and Purification of \u3cem\u3eVibrio proteolyticus (Aeromonas proteolytica)\u3c/em\u3e Aminopeptidase: A Rapid Protocol

Metalloaminopeptidases (mAPs) are enzymes that are involved in HIV infectivity, tumor growth and metastasis, angiogenesis, and bacterial infection. Investigation of structure–function relationships in mAPs is a prerequisite to rational design of anti-mAP chemotherapeutics. The most intensively studied member of the biomedically important dinuclear mAPs is the prototypical secreted Vibrio proteolyticus di-zinc aminopeptidase (VpAP). The wild-type enzyme is readily purified from the supernatant of cultures of V. proteolyticus, but recombinant variants require expression in Escherichia coli. A greatly improved system for the purification of recombinant VpAP is described. A VpAP-(His)6 polypeptide, containing an N-terminal propeptide, and a C-terminal (His)6 adduct, was purified by metal ion affinity chromatography from the supernatant of cultures of E. coli. This single step replaced the sequence of (NH4)2SO4 fractionation, and anion-exchange and hydrophobic interaction chromatographic separations of earlier methods. Traditionally, recombinant VpAP proenzyme has been treated with proteinase K and with heat (70 °C), to remove the N- and C-terminal regions, and yield the mature active enzyme. This method is unsuitable for VpAP variants that are unstable towards these treatments. In the new method, the hitherto noted, but not fully appreciated, ability of VpAP to autocatalyze the hydrolysis of the N-terminal propeptide and C-terminal regions was exploited; extensive dialysis of the highly purified VpAP-(His)6 full-length polypeptide yielded the mature active protein without recourse to proteinase K or heat treatment. Purification of variants that have previously defied isolation as mature forms of the protein was thus carried out

EPR of Co(II) as a Structural and Mechanistic Probe of Metalloprotein Active Sites: A Review of Studies on Aminopeptidase

Co(II) can often be substituted for Zn(II) in zinc-dependent metalloenzymes to provide spectroscopically accessible forms of the enzymes. Co(II) is an excellent spectroscopic probe as it is both optically active and EPR active. Further, its fast relaxation properties make it a useful paramagnetic shift reagent in NMR. In EPR, the dependence of the spectra of high-spin Co(II) on E/D and the sensitivity of the resolvability of the 59Co hyperfine structure to strain terms allow structural information to be inferred from the EPR spectra. In addition to its useful spectroscopic properties, Co(II) is often an extremely good functional mimic of Zn(II), and Co(II)-substituted zinc-dependent enzymes often display catalytic activities analogous to the native Zn(II)-containing enzyme forms. It is therefore somewhat surprising that there are few examples of EPR studies of Co(II)-substituted enzymes. The most detailed studies carried out to date are those on the aminopeptidase from Aeromonas proteolytica. Therefore, the methodology of extracting structural information from EPR of Co(II)-containing proteins is described using studies on A. proteolytica aminopeptidase as an example

Spin Hamiltonian Parameters for Cu(II)−Prion Peptide Complexes from L-Band Electron Paramagnetic Resonance Spectroscopy

Cu(II) is an essential element for life but is also associated with numerous and serious medical conditions, particularly neurodegeneration. Structural modeling of crystallization-resistant biological Cu(II) species relies on detailed spectroscopic analysis. Electron paramagnetic resonance (EPR) can, in principle, provide spin Hamiltonian parameters that contain information on the geometry and ligand atom complement of Cu(II). Unfortunately, EPR spectra of Cu(II) recorded at the traditional X-band frequency are complicated by (i) strains in the region of the spectrum corresponding to the g∥ orientation and (ii) potentially very many overlapping transitions in the g⊥ region. The rapid progress of density functional theory computation as a means to correlate EPR and structure, and the increasing need to study Cu(II) associated with biomolecules in more biologically and biomedically relevant environments such as cells and tissue, have spurred the development of a technique for the extraction of a more complete set of spin Hamiltonian parameters that is relatively straightforward and widely applicable. EPR at L-band (1−2 GHz) provides much enhanced spectral resolution and straightforward analysis via computer simulation methods. Herein, the anisotropic spin Hamiltonian parameters and the nitrogen coordination numbers for two hitherto incompletely characterized Cu(II)-bound species of a prion peptide complex are determined by analysis of their L-band EPR spectra

EPR Studies on the Mono- and Dicobalt(II)-Substituted Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e. Insight into the Catalytic Mechanism of Dinuclear Hydrolases

The structure and function of the prototypical dinuclear hydrolase, namely, the aminopeptidase from Aeromonas proteolytica (AAP), was probed by EPR spectroscopy of the mono- and dicobalt(II)-substituted derivatives. A new systematic protocol for the interpretation of Co(II) EPR spectra is described and the S = 3/2 spin states of the Co(II)-substituted forms of the enzyme have been characterized. This protocol allows the simulation of line shape using theoretically allowed geff values corresponding to an isotropic greal value. In addition, the gross distortion of EPR spectra of high-spin S = 3/2 Co(II) ions has been investigated, and the effects of saturation on the line shapes and on simulation-derived spectral parameters are discussed. For [Co-(AAP)], a distinctive EPR signal was observed in which the hyperfine pattern due to 59Co was not centered on the low-field absorption feature, and the signal could not be simulated as a single species. Subtraction of EPR spectra recorded at different temperatures revealed that two species were, in fact, present in samples of [Co-(AAP)]. The first species was a relatively featureless axial signal with geff values of 5.75, 4.50, and 2.50. These values correspond to an Ms = |±1/2〉 ground-state transition with greal = 2.57 and E/D = 0.08. The second species had geff values of 6.83, 2.95, and 1.96 and exhibited a characteristic eight-line 59Co hyperfine pattern with Az = 7.2 mT. The observed 59Co hyperfine lines were simulated in both line width as well as signal intensity for the first time. These parameters correspond to the Ms = |±1/2〉 ground-state transition with greal = 2.57; however, the signal exhibited marked rhombicity (E/D = 0.28), consistent with a highly distorted tetrahedral Co(II) species. The possibility that the spectrum could be due to contributions from the Ms = |±1/2〉 and Ms = |±3/2〉 doublets of a single spin system was investigated, but subtraction of spectra recorded at various temperatures clearly indicated that the features at g = 2.95 and g = 1.96 were correlated with the feature at g = 6.83. In addition, at temperatures above 15 K, the signal intensity rapidly decreases and the signal is lost. The EPR spectrum of [CoCo(AAP)] reveals a relatively featureless signal that was simulated as a single species with geff(1,2,3) values of 5.10, 3.85, and 2.19; Ms = |±1/2〉; greal = 2.25; E/D = 0.095. The intensity of the observed signal for [CoCo(AAP)] corresponded to 0.13 spin/mol of Co(II). These data strongly suggest that the two Co(II) ions in the active site of AAP experience significant spin−spin interaction and are either antiferromagnetically or ferromagnetically coupled. Perpendicular mode EPR titration of apo-AAP with Co(II) revealed a low-field signal extending out of zero-field in samples with more than 1 equiv of Co(II) added. This type of EPR absorption is indicative of an integral spin system. Coincident with the appearance of the low-field perpendicular mode signal was the appearance of a parallel mode EPR signal with g ∼ 12. These data represent the first definitive evidence for ferromagnetic coupling between two high-spin S = 3/2 Co(II) ions in a dinuclear center. The effect of pH, added peroxide, and the coordination of the competitive inhibitor 1-butaneboronic acid (BuBA) on the signal both confirm the origin of the signal and provide important mechanistic information for this novel dicobalt(II) active site cluster. Based on the present study and the available literature data, a detailed mechanism of action is proposed for AAP

Spectroscopically Distinct Cobalt(II) Sites in Heterodimetallic Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e: Characterization of Substrate Binding

The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and 5.23 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with geff values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line 59Co hyperfine pattern with an Az(59Co) of 7.0 mT. These parameters correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion. Summation of these two species provided an excellent simulation of the observed [CoZn(AAP)] EPR spectrum. The EPR spectrum of [ZnCo(AAP)] also contained two species, at least one of which also exhibited 59Co hyperfine features. However, this signal exhibited little pH dependence, and individual species could not be isolated. The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to [CoZn(AAP)] resulted in a distinct change in the EPR spectrum; however, addition of BuBA to [ZnCo(AAP)] left the EPR spectrum completely unperturbed. These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site. On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322−5331]. From difference spectroscopy and from the simulation of the [CoZn(AAP)] EPR spectrum, a third signal appearing upon BuBA binding was isolated. This signal was simulated with geff values of 2.08, 3.15, and 6.15 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved 59Co hyperfine pattern with an Az(59Co) value of 4.0 mT. Summation of the these three species provided an excellent simulation of the observed [CoZn(AAP)] + BuBA EPR spectrum at both pH values. This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP [Bennett, B., & Holz, R. C. (1997) J. Am. Chem. Soc. 119, 1923−1933; Chen, G., et al. (1997) Biochemistry 36, 4278−4286]

EPR Methods for Biological Cu(II): L-Band CW and NARS

Abstract: Copper has many roles in biology that involve the change of coordination sphere and/or oxidation state of the copper ion. Consequently, the study of copper in heterogeneous environments is an important area in biophysics. EPR is a primary technique for the investigation of paramagnetic copper, which is usually the isolated Cu(II) ion, but sometimes as Cu(II) in different oxidation states of multitransition ion clusters. The gross geometry of the coordination environment of Cu(II) can often be determined from a simple inspection of the EPR spectrum, recorded in the traditional X-band frequency range (9–10 GHz). Identification and quantitation of the coordinating ligand atoms, however, is not so straightforward. In particular, analysis of the superhyperfine structure on the EPR spectrum, to determine the number of coordinated nitrogen atoms, is fraught with difficulty at X-band, despite the observation that the overwhelming number of EPR studies of Cu(II) in the literature have been carried out at X-band. Greater reliability has been demonstrated at S-band (3–4 GHz), using the low-field parallel (gz) features. However, analysis relies on clear identification of the outermost superhyperfine line, which has the lowest intensity of all the spectral features. Computer simulations have subsequently indicated that the much more intense perpendicular region of the spectrum can be reliably interpreted at L-band (2 GHz). The present work describes the development of L-band EPR of Cu(II) into a routine method that is applicable to biological samples

A Cobalt-Containing Eukaryotic Nitrile Hydratase

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2 homodimer in solution, analogous to the α2β2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds

Biocompatible Copper Oxide Nanoparticle Composites from Cellulose and Chitosan: Facile Synthesis, Unique Structure, and Antimicrobial Activity

Copper in various forms has been known to have bactericidal activity. Challenges to its application include preventing mobilization of the copper, to both extend activity and avoid toxicity, and bioincompatibility of many candidate substrates for copper immobilization. Using a simple ionic liquid, butylmethylimmidazolium chloride as the solvent, we developed a facile and green method to synthesize biocompatible composites containing copper oxide nanoparticles (CuONPs) from cellulose (CEL) and chitosan (CS) or CEL and keratin (KER). Spectroscopy and imaging results indicate that CEL, CS, and KER remained chemically intact and were homogeneously distributed in the composites with CuONPs with size of 22 ± 1 nm. Electron paramagnetic resonance (EPR) suggests that some 25% of the EPR-detectable Cu(II) is present as a monomeric species, chemically anchored to the substrate by two or more nitrogen atoms, and, further, adopts a unique spatially oriented conformation when incorporated into the [CEL + CS] composite but not in the [CEL + KER] composite. The remaining 75% of EPR-detectable Cu(II) exhibited extensive spin–spin interactions, consistent with Cu(II) aggregates and Cu(II) on the surface of CuONPs. At higher levels of added copper (\u3e59 nmol/mg), the additional copper was EPR-silent, suggesting an additional phase in larger CuONPs, in which S \u3e 0 spin states are either thermally inaccessible or very fast-relaxing. These data suggest that Cu(II) initially binds substrate via nitrogen atoms, from which CuONPs develop through aggregation of copper. The composites exhibited excellent antimicrobial activity against a wide range of bacteria and fungi, including methicillin-resistant Staphylococcus aureus; vancomycin-resistant Enterococcus; and highly resistant Escherichia coli, Streptococcus agalactiae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Candida albicans. Expectedly, the antibacterial activity was found to be correlated with the CuONPs content in the composites. More importantly, at CuONP concentration of 35 nmol/mg or lower, bactericidal activity of the composite was complemented by its biocompatibility with human fibroblasts