Comparison of metal‐bound and unbound structures of aminopeptidase B proteins from Escherichia coli and Yersinia pestis

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal‐dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn²⁺ and Mn²⁺, while the second structure has two Zn²⁺ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N‐terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C‐terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.     

Crystal Structure of the Leucine Aminopeptidase from Pseudomonas putida Reveals the Molecular Basis for its Enantioselectivity and Broad Substrate Specificity

The zinc-dependent leucine aminopeptidase from Pseudomonas putida (ppLAP) is an important enzyme for the industrial production of enantiomerically pure amino acids. To provide a better understanding of its structure-function relationships, the enzyme was studied by X-ray crystallography. Crystal structures of native ppLAP at pH 9.5 and pH 5.2, and in complex with the inhibitor bestatin, show that the overall folding and hexameric organization of ppLAP are very similar to those of the closely related di-zinc leucine aminopeptidases (LAPs) from bovine lens and Escherichia coli. At pH 9.5, the active site contains two metal ions, one identified as Mn²⁺ or Zn²⁺ (site 1), and the other as Zn²⁺ (site 2). By using a metal-dependent activity assay it was shown that site 1 in heterologously expressed ppLAP is occupied mainly by Mn²⁺. Moreover, it was shown that Mn²⁺ has a significant activation effect when bound to site 1 of ppLAP. At pH 5.2, the active site of ppLAP is highly disordered and the two metal ions are absent, most probably due to full protonation of one of the metal-interacting residues, Lys267, explaining why ppLAP is inactive at low pH. A structural comparison of the ppLAP-bestatin complex with inhibitor-bound complexes of bovine lens LAP, along with substrate modelling, gave clear and new insights into its substrate specificity and high level of enantioselectivity.     

Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys¹⁴⁷, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys¹⁴⁷ to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca²⁺ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.     

Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-pNA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat_H-Ala-pNA L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.     

Identification of histidine residues important in the catalysis and structure of aspartyl aminopeptidase

Aspartyl aminopeptidase (DAP), a widely distributed and abundant cytosolic enzyme, removes glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides. DAP is a member of the M18 family of the MH clan of cocatalytic metallopeptidases. The human and mouse enzymes have been cloned. We have identified 8 highly homologous eukaryotic sequences that are probable aspartyl aminopeptidases. Eight histidine residues of human DAP were sequentially mutated to phenylalanine. Mutation of His94, His170, and His440 abolished enzymatic activity. His94 and His440 are postulated to be involved in binding cocatalytic zinc atoms by homology with other members of the MH clan. Mutation of His352 dramatically reduced enzyme activity. Gel-filtration analysis of the His352 mutant revealed destabilization of the quaternary structure and dissociation of the native 440-kDa enzyme. Mutation of His33 and of histidines residing in a cluster at residues 349, 359, and 363 all decreased k(cat). These studies reveal an important role for histidine residues both in catalysis and in the structural integrity of DAP.     

Leucine aminopeptidase in exsheathing fluid of North American and Australian Haemonchus contortus

Rogers W. P. and Brooks F. 1978. Leucine aminopeptidase in exsheathing fluid of north American and Australian Haemonchus contortus. International Journal for Parasitology8: 55–58. Juveniles of Haemonchus contortus from north America and Australia produced exsheathing fluid containing leucine aminopeptidase when stimulated in tetraborate-carbon dioxide medium. Exsheathment in this medium was inhibited by 1, 10-phenanthroline, 10^?3M, and this inhibition was largely reversed by Zn²⁺, 10^?3M. This supports the view that the enzyme is produced by the juveniles and that it is concerned in exsheathment.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin

A membrane-bound aminopeptidase which cleaves the tyrosin-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a K_m value of 35 μM and also hydrolyzed basic, neutral and aromatic amino acid β-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co²⁺, Mg²⁺ and Zn²⁺. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a K_i value of 0.02 μM.     

Peptidases from pig reproductive tract: purification and properties of aminopeptidases from uterine secretions, allantoic fluid, and amniotic fluid

In ovariectomized sows, aminopeptidase is secreted into the uterine lumen under the influence of progesterone. The enzyme also accumulates in allantoic and amniotic fluids of pregnant animals. We have purified the predominant form of this enzyme from uterine flushings, allantoic fluid, and amniotic fluid by the following steps: ammonium sulfate precipitation, Sepharose 6B chromatography, ion-exhange chromatography on diethylaminoethyl cellulose, and affinity chromatography usingl-leucylglycine immobilized on agarose. The overall procedure gave approximately 974-, 110-, and 230-fold purifications of the allantoic, uterine, and amniotic enzymes, respectively. The enzymes from all three sources are glycoproteins with pI's around 4 and molecular weights of about 480,000. They may be dissociated into six apparently identical subunits of molecular weight 80,000 as judged by sodium dodecyl sulfate gel electrophoresis. With l-leucyl-β-naphthylamide as substrate the pH optimum and apparent K_m value for each enzyme were 7.1 and 14 μm, respectively. However, the uterine and allantoic aminopeptidases exhibited V values of 0.35 μmol of substrate hydrolyzed/min/mg of protein, whereas the V for the amniotic enzyme was at least sixfold greater. The amniotic enzyme also differed from the other two in pH and temperature stability. The activity of all three enzymes was stimulated by Co²⁺ and inhibited by Cu²⁺, Fe³⁺, and chelating agents, while iodoacetate and mercaptoethanol had no effect on catalysis. The effect of Co²⁺ on the allantoic enzyme was investigated in further detail. The stimulation of peptidase activity by Co²⁺ was shown to be a complex process but consistent with Co²⁺ replacing another metal at the active site and at some other additional site on the enzyme. The function of the aminopeptidases in the pregnant uterus is unknown.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

The peptidase activity of human serum butyrylcholinesterase: Studies using monoclonal antibodies and characterization of the peptidase

Purified human serum butyrylcholinesterase, which exhibits cholinesterase, aryl acylamidase, and peptidase activities, was cross-reacted with two different monoclonal antibodies raised against human serum butyrylcholinesterase. All three activities were immunoprecipitable at different dilutions of the two monoclonal antibodies. At the highest concentration of the antibodies used, nearly 100% of all three activities were precipitated, and could be recovered to 90–95% in the immunoprecipitate. The peptidase activity exhibited by the purified butyryl-cholinesterase was further characterized using both Phe-Leu and Leu-enkephalin as substrates. ThepH optimum of the peptidase was in the range of 7.5–9.5 and the divalent cations Co²⁺, Mn²⁺, and Zn²⁺ stimulated its activity. EDTA and other metal complexing agents inhibited its activity. Thiol agents and -SH group modifiers had no effect. The serine protease inhibitors, diisopropylfluorophosphate and phenyl methyl sulfonyl fluoride, did not inhibit. When histidine residues in the enzyme were modified by diethylpyrocarbonate, the peptidase activity was not affected, but the stimulatory effect of Co²⁺, Mn²⁺, and Zn²⁺ disappeared, suggesting the involvement of histidine residues in metal ion binding. These general characteristics of the peptidase activity were also exhibited by a 50 kD fragment obtained by limited -chymotrypsin digestion of purified butyrylcholinesterase. Under all assay conditions, the peptidase released the two amino acids, leucine and phenylalanine, from the carboxy terminus of Leu-enkephalin as verified by paper chromatography and HPLC analysis. The results suggested that the peptidase behaved like a serine, cysteine, thiol-independent metallopeptidase.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

ZnuA is the periplasmic Zn²⁺-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn²⁺-bound, and Co²⁺-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn²⁺ with Co²⁺ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn²⁺ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn²⁺ (estimated K _d < 20 nM), Co²⁺, Ni²⁺, Cu²⁺, Cu⁺, and Cd²⁺, but not Mn²⁺. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn²⁺ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bacillus subtilis aminopeptidase: purification, characterization and some enzymatic properties.

The extracellular aminopeptidase from Bacillus subtilis was purified 300-fold by a simple procedure which gave a high recovery of enzyme. The native enzyme was shown to be a monomer of molecular weight 46,500 and to contain 1 g-atom of Zn²⁺ per mole of protein. Amino acid analyses demonstrated the protein to be rich in acidic residues and Lys, to possess about 3 residues of Met, and to be devoid of Cys. When activated with 5 mm Co(NO₃)₂ for 90 min the activity of the native enzyme was increased; the amount of activation depended on the identity of the substrate. Cobalt activation involved the reversible binding of 1 g-atom of Co²⁺ per mole of protein, without displacing the native Zn²⁺; K_Co was 1.25 mm. Zinc ions competed with Co²⁺ during activation, a process characterized by a _KZn of 28 μm. Ions other than Co²⁺ did not appreciably activate the enzyme.     

Separating the role of protein restraints and local metal-site interaction chemistry in the thermodynamics of a zinc finger protein

We express the effective Hamiltonian of an ion-binding site in a protein as a combination of the Hamiltonian of the ion-bound site in vacuum and the restraints of the protein on the site. The protein restraints are described by the quadratic elastic network model. The Hamiltonian of the ion-bound site in vacuum is approximated as a generalized Hessian around the minimum energy configuration. The resultant of the two quadratic Hamiltonians is cast into a pure quadratic form. In the canonical ensemble, the quadratic nature of the resultant Hamiltonian allows us to express analytically the excess free energy, enthalpy, and entropy of ion binding to the protein. The analytical expressions allow us to separate the roles of the dynamic restraints imposed by the protein on the binding site and the temperature-independent chemical effects in metal-ligand coordination. For the consensus zinc-finger peptide, relative to the aqueous phase, the calculated free energy of exchanging Zn²⁺ with Fe²⁺, Co²⁺, Ni²⁺, and Cd²⁺ are in agreement with experiments. The predicted excess enthalpy of ion exchange between Zn²⁺ and Co²⁺ also agrees with the available experimental estimate. The free energy of applying the protein restraints reveals that relative to Zn²⁺, the Co²⁺, and Cd²⁺-site clusters are more destabilized by the protein restraints. This leads to an experimentally testable hypothesis that a tetrahedral metal binding site with minimal protein restraints will be less selective for Zn²⁺ over Co²⁺ and Cd²⁺ compared to a zinc finger peptide. No appreciable change is expected for Fe²⁺ and Ni²⁺. The framework presented here may prove useful in protein engineering to tune metal selectivity.     

A Zinc Site in the C-Terminal Domain of RAG1 Is Essential for DNA Cleavage Activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn²⁺ ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn²⁺ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn²⁺ ions. Stripping the full complement of bound Zn²⁺ ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn²⁺ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn²⁺ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn²⁺ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn²⁺ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn²⁺ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.     

Coordination contributions to protein stability in metal-substituted carbonic anhydrase

Contributions of the active site metal to the stability of carbonic anhydrase (CA) were quantified by differential scanning calorimetry and complementary unfolding measurements of CA substituted with Co²⁺, Cd²⁺, Cu²⁺, Ni²⁺ and Mn²⁺. The metal ions stabilize the protein to different extent, with the highest stability provided by the native Zn²⁺. This additional stability does not correlate with the enthalpy of the three metal-imidazole (His) bonds at the active site or other properties of the metal ions (charge density, hydration enthalpy). However, DFT calculations reveal an energetic penalty associated with metal coordination at the active site, and the magnitude of this penalty correlates inversely with metal contributions to the stability of the protein. While the affinity of CA for metal ions generally reflects the Irving–Williams series, the additional thermal stability provided by metal ions is modulated by the rigid His₃ coordination that is imposed at the protein site.     

On the magnetic properties of cobalt substituted bovine superoxide dismutase derivatives.

We have measured the magnetic susceptibility in the temperature range 1.4–77°K of three derivatives of bovine superoxide dismutase in which Co²⁺ was substituted for Zn²⁺: (1) 2Co²⁺ — in which Co²⁺ binds to the normal Zn²⁺ site and the Cu²⁺ site is unoccupied, (2) 2Co²⁺2Cu²⁺ — in which the Zn²⁺ site is occupied by Co²⁺ and the copper sites contains Cu²⁺ and (3) 2Co²⁺2Cu⁺ — which is the reduced form of the second derivative. The 2Co²⁺ protein exhibits Curie paramagnetism indicating $\text{S′}\text{=}\text{1}\text{2}$ and the zero-field splitting must be greater than ?20 cm^?1. The same propeties have been observed with the 2Co²⁺2Cu⁺-protein. By contrast, the 2Co²⁺2Cu²⁺-derivative exhibits relatively little paramagnetism, some of which arises from non-specifically associated metal ions. The lower susceptibility is due to antiferromagnetic coupling between Co²⁺ and Cu²⁺, and the magnitude of the coupling constant is probably ?5 cm^?1.     

Structural insights to the metal specificity of an archaeal member of the LigD 3'-phosphoesterase DNA repair enzyme family

LigD 3′-phosphoesterase (PE) enzymes perform end-healing reactions at DNA breaks. Here we characterize the 3′-ribonucleoside-resecting activity of Candidatus Korarchaeum PE. CkoPE prefers a single-stranded substrate versus a primer–template. Activity is abolished by vanadate (10 mM), but is less sensitive to phosphate (IC₅₀ 50 mM) or chloride (IC₅₀ 150 mM). The metal requirement is satisfied by manganese, cobalt, copper or cadmium, but not magnesium, calcium, nickel or zinc. Insights to CkoPE metal specificity were gained by solving new 1.5 Å crystal structures of CkoPE in complexes with Co²⁺ and Zn²⁺. His9, His15 and Asp17 coordinate cobalt in an octahedral complex that includes a phosphate anion, which is in turn coordinated by Arg19 and His51. The cobalt and phosphate positions and the atomic contacts in the active site are virtually identical to those in the CkoPE·Mn²⁺ structure. By contrast, Zn²⁺ binds in the active site in a tetrahedral complex, wherein the position, orientation and atomic contacts of the phosphate are shifted and its interaction with His51 is lost. We conclude that: (i) PE selectively binds to ‘soft’ metals in either productive or non-productive modes and (ii) PE catalysis depends acutely on proper metal and scissile phosphate geometry.