Peptidylglycine Α-hydroxylating Monooxygenase Research Articles

Reaction Mechanism of the Bicopper Enzyme Peptidylglycine α-Hydroxylating Monooxygenase

Peptidylglycine α-hydroxylating monooxygenase is a noninteracting bicopper enzyme that stereospecifically hydroxylates the terminal glycine of small peptides for its later amidation. Neuroendocrine messengers, such as oxytocin, rely on the biological activity of this enzyme. Each catalytic turnover requires one oxygen molecule, two protons from the solvent, and two electrons. Despite this enzyme having been widely studied, a consensus on the reaction mechanism has not yet been found. Experiments and theoretical studies favor a pro-S abstraction of a hydrogen atom followed by the rebinding of an OH group. However, several hydrogen-abstracting species have been postulated; because two protons are consumed during the reaction, several protonation states are available. An electron transfer between the copper atoms could play a crucial role for the catalysis as well. This leads to six possible abstracting species. In this study, we compare them on equal footing. We perform quantum mechanics/molecular mechanics calculations, considering the glycine hydrogen abstraction. Our results suggest that the most likely mechanism is a protonation of the abstracting species before the hydrogen abstraction and another protonation as well as a reduction before OH rebinding.

Investigation of the Hydroxylation Mechanism of Noncoupled Copper Oxygenases by Ab Initio Molecular Dynamics Simulations

In Nature, the family of copper monooxygenases comprised of peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM) is known to perform dioxygen-dependent hydroxylation of aliphatic C-H bonds by using two uncoupled metal sites. In spite of many investigations, including biochemical, chemical, and computational, details of the C-H bond oxygenation mechanism remain elusive. Herein we report an investigation of the mechanism of hydroxylation by PHM by using hybrid quantum/classical potentials (i.e., QM/MM). Although previous investigations using hybrid QM/MM techniques were restricted to geometry optimizations, we have carried out ab initio molecular dynamics simulations in order to include the intrinsic flexibility of the active sites in the modeling protocol. The major finding of this study is an extremely fast rebound step after the initial hydrogen-abstraction step promoted by the cupric-superoxide adduct. The hydrogen-abstraction/rebound sequence leads to the formation of an alkyl hydroperoxide intermediate. Long-range electron transfer from the remote copper site subsequently triggers its reduction to the hydroxylated substrate. We finally show two reactivity consequences inherent in the new mechanistic proposal, the investigation of which would provide a means to check its validity by experimental means.

De Novo-Designed Metallopeptides with Type 2 Copper Centers: Modulation of Reduction Potentials and Nitrite Reductase Activities

Enzymatic reactions involving redox processes are highly sensitive to the local electrostatic environment. Despite considerable effort, the complex interactions among different influential factors in native proteins impede progress toward complete understanding of the structure-function relationship. Of particular interest is the type 2 copper center Cu(His)3, which may act as an electron transfer center in peptidylglycine α-hydroxylating monooxygenase (PHM) or a catalytic center in copper nitrite reductase (CuNiR). A de novo design strategy is used to probe the effect of modifying charged amino acid residues around, but not directly bound to, a Cu(His)3 center embedded in three-stranded coiled coils (TRI-H)3 [TRI-H = Ac-G WKALEEK LKALEEK LKALEEK HKALEEK G-NH2]. Specifically, the peptide TRI-EH (=TRI-HK22E) alters an important lysine to glutamate just above the copper binding center. With a series of TRI-EH peptides mutated below the metal center, we use a variety of spectroscopies (EPR, UV-vis, XAS) to show a direct impact on the protonation equilibria, copper binding affinities, reduction potentials, and nitrite reductase activities of these copper-peptide complexes. The potentials at a specific pH vary by 100 mV, and the nitrite reductase activities range over a factor of 4 in rates. We also observe that the affinities, potentials, and catalytic activities are strongly influenced by the pH conditions (pH 5.8-7.4). In general, Cu(II) affinities for the peptides are diminished at low pH values. The interplay among these factors can lead to a 200 mV shift in reduction potential across these peptides, which is determined by the pH-dependent affinities of copper in both oxidation states. This study illustrates the strength of de novo protein design in elucidating the influence of ionizable residues on a particular redox system, an important step toward understanding the factors that govern the properties of this metalloenzyme with a goal of eventually improving the catalytic activity.

Redox Properties of a Mononuclear Copper(II)-Superoxide Complex

Redox properties of a mononuclear copper(II) superoxide complex, (L)Cu(II)-OO(•), supported by a tridentate ligand (L = 1-(2-phenethyl)-5-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane) have been examined as a model compound of the putative reactive intermediate of peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) (Kunishita et al. J. Am. Chem. Soc. 2009, 131, 2788-2789; Inorg. Chem. 2012, 51, 9465-9480). On the basis of the reactivity toward a series of one-electron reductants, the reduction potential of (L)Cu(II)-OO(•) was estimated to be 0.19 ± 0.07 V vs SCE in acetone at 298 K (cf. Tahsini et al. Chem.-Eur. J. 2012, 18, 1084-1093). In the reaction of TEMPO-H (2,2,6,6-tetramethylpiperidine-N-hydroxide), a simple HAT (hydrogen atom transfer) reaction took place to give the corresponding hydroperoxide complex LCu(II)-OOH, whereas the reaction with phenol derivatives ((X)ArOH) gave the corresponding phenolate adducts, LCu(II)-O(X)Ar, presumably via an acid-base reaction between the superoxide ligand and the phenols. The reaction of (L)Cu(II)-OO(•) with a series of triphenylphosphine derivatives gave the corresponding triphenylphosphine oxides via an electrophilic ionic substitution mechanism with a Hammett ρ value as -4.3, whereas the reaction with thioanisole (sulfide) only gave a copper(I) complex. These reactivities of (L)Cu(II)-OO(•) are different from those of a similar end-on superoxide copper(II) complex supported by a tetradentate TMG3tren ligand (1,1,1-Tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine (Maiti et al. Angew. Chem., Int. Ed. 2008, 47, 82-85).

Transcript response of soft coral (Scleronephthya gracillimum) on exposure to polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are the most persistent organic pollutants in worldwide aquatic environments. The extensive isolation of genes responsive to PAH pollution in soft coral (Scleronephthya gracillimum) is described herein. Soft coral colonies were exposed to 100μg/L of a standard mixture of PAHs. Gene candidates with transcript levels that changed in response to PAH exposure were identified by differential display polymerase chain reaction (DD-PCR). There were 37 types of candidate genes identified, of which 20 were upregulated in expression and 17 were downregulated. The functions of the genes identified included oxidative stress response, ribosomal structure maintenance, molecular chaperone activity, protein kinase activation and tumorigenesis, defense mechanisms, transcription, and other biological responses. mRNA quantification was carried out using real-time quantitative PCR in eight selected genes: cytosolic malate dehydrogenase, protein disulfide isomerase, ribosomal protein L6, ral guanine nucleotide dissociation stimulator-like 1, poly(ADP-ribose) polymerase 4, peptidylglycine α-hydroxylating monooxygenase, a disintegrin and metalloproteinase (ADAM) metallopeptidase protein, and eukaryotic initiation factor 4 gamma 3. Changes in transcript levels were consistent with DD-PCR results. The gene candidates isolated in this study were differentially expressed and therefore have potential as molecular biomarkers for understanding coral responses to environmental stressors.

Coordination of peroxide to the Cu(M) center of peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study.

Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine α-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction-the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. The hydroxylation reaction is copper- and O(2)-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests that O(2) is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (Cu(H) and Cu(M)). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-Å-resolution structure (hydro)peroxide binds solely to Cu(M) in a slightly asymmetric side-on mode. The O-O interatomic distance of the copper-bound ligand is 1.5 Å, characteristic of peroxide/hydroperoxide species, and the Cu-O distances are 2.0 and 2.1 Å. Density functional theory calculations using the first coordination sphere of the Cu(M) active site as a model system show that the computed energies of the side-on L(3)Cu(M)(II)-O(2) (2-) species and its isomeric, end-on structure L(3)Cu(M)(I)-O(2) (·-) are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L(3)Cu(M)(II)O(2) (2-) is in good agreement with the results of a hybrid quantum mechanical-molecular mechanical optimization of this species.

Active Site Models for the CuA Site of Peptidylglycine α-Hydroxylating Monooxygenase and Dopamine β-Monooxygenase

A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated product. The present results have indicated that a superoxo species having a four-coordinate distorted tetrahedral geometry could be reactive enough to induce the direct C-H bond activation of aliphatic substrates in the enzymatic systems.

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors.

A "Neural" Enzyme in Nonbilaterian Animals and Algae: Preneural Origins for Peptidylglycine -Amidating Monooxygenase

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.

Coordination of Hydrogen Peroxide by a Copper Center in Peptidylglycine Alpha-Hydroxylating Monooxygenase (PHM). Structural and Computational Study

Many bioactive peptides, such as hormones and growth factors require amidation of the C-terminus for their full biological activity. The enzyme peptidylglycine α-hydroxylating monooxygenase (PHM) carries out the first step of the amidation process, the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. Two non-equivalent copper sites in PHM (CuH and CuM) play distinct roles in the reaction cycle: CuM serves as an oxygen activation and hydrogen abstraction site, while CuH is involved in electron transfer. The proposed mechanism suggests that dioxygen is activated through a two-electron reduction, where each of the copper centers provides a single electron. However, there is an ambiguity regarding the characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate. To further investigate the nature of the key intermediates in the PHM cycle we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.95 A resolution structure, the (hydro)peroxide ligand coordinates solely to CuM in a slightly asymmetric side-on mode. The copper-oxygen distances are 1.9 and 2.1 A and the O-O bond 1.5 A. The interatomic O-O distance is characteristic of peroxide/hydroperoxide species, and is significantly longer than the distance typically observed in the superoxide molecule. In addition to the x-ray diffraction studies, we performed DFT calculations using the first coordination sphere of the CuM active site as a model system. Comparison of the relative energies associated with side-on Cu(II)-O22- species and its resonant, end-on structure Cu(I)-O2•- suggests that these two intermediates are significantly populated within the protein environment; this observation has important mechanistic implications.

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM+/−) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM+/− mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM+/− mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.

Evidence for Substrate Preorganization in the Peptidylglycine α-Amidating Monooxygenase Reaction Describing the Contribution of Ground State Structure to Hydrogen Tunneling

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the corresponding bioactive α-amidated peptide hormone. This conversion involves two sequential reactions both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-, and O(2)-dependent stereospecific hydroxylation at the α-carbon of the C-terminal glycine, is catalyzed by peptidylglycine α-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation of the carbinolamide intermediate, is catalyzed by peptidylglycine amidoglycolate lyase. Quantum mechanical tunneling dominates PHM-dependent C(α)-H bond activation. This study probes the substrate structure dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy perturbation, and equilibrium molecular dynamics were used to study the role played by ground-state substrate structure on PHM catalysis. Our data show that all Ν-acylglycines bind sequentially to PHM in an equilibrium-ordered fashion. The primary deuterium KIE displays a linear decrease with respect to acyl chain length for straight-chain N-acylglycine substrates. Docking orientation of these substrates displayed increased dissociation energy proportional to hydrophobic pocket interaction. The decrease in KIE with hydrophobicity was attributed to a preorganization event which decreased reorganization energy by decreasing the conformational sampling associated with ground state substrate binding. This is the first example of preorganization in the family of noncoupled copper monooxygenases.

Differential Reactivity between Two Copper Sites in Peptidylglycine α-Hydroxylating Monooxygenase

Peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the Cα of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu(M) and Cu(H)) separated by ∼11 Å. In the resting state of the enzyme, Cu(M) is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu(H) has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu(M) is identified as the metal involved in catalysis, while Cu(H) is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO(2)(-)), azide (N(3)(-)), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO(2), shows that nitrite anion coordinates Cu(M) in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu(H), despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu(M) but not Cu(H) in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN(3) and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu(H) is also observed in the reduced form of the enzyme: CO binds Cu(M) but not Cu(H) in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5 mM ascorbic acid (reductant). The necessity of Cu(H) to maintain its redox potential in a narrow range compatible with its role as an electron-transfer site seems to explain the lack of coordination of small molecules to Cu(H); coordination of any external ligand will certainly modify its redox potential.

A PAL for Schistosoma mansoni PHM

Parasitic helminth neuromuscular function is a proven target for chemotherapeutic control. Although neuropeptide signalling plays a key role in helminth motor function, it has not yet provided targets for known anthelmintics. The majority of biologically active neuropeptides display a C-terminal amide (NH2) motif, generated exclusively by the sequential action of two enzymes, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylglycine α-amidating lyase (PAL). Further to our previous description of a monofunctional PHM enzyme (SmPHM) from the human blood fluke Schistosoma mansoni, here we describe a cDNA encoding S. mansoni PAL (SmPAL). SmPAL is a monofunctional enzyme which, following heterologous expression, we find to have functionally similar catalytic activity and optimal pH values, but key catalytic core amino acid substitutions, when compared to other known PALs including those found in humans. We have used in situ hybridisation to demonstrate that in adult schistosomes, SmPAL mRNA (Sm-pal-1) is expressed in neuronal cell bodies of the central nervous system, consistent with a role for amidated neuropeptides in S. mansoni neuromuscular function. In order to validate SmPAL as a putative drug target we applied published RNA interference (RNAi) methods in efforts to trigger knockdown of Sm-pal-1 transcript in larval schistosomula. Although transcript knockdown was recorded on several occasions, silencing was variable and inconsistent and did not associate with any observable aberrant phenotype. The inconsistent outcomes of RNAi suggest that there may be tissue-specific differences in the applicability of RNAi methods for S. mansoni, with neuronal targets proving more difficult or refractory to knockdown. The key role played by schistosome amidating enzymes in neuropeptide maturation make them appealing as drug targets; their validation as such will depend on the development of more robust reverse genetic tools to facilitate efficient neuronal gene function studies.

Dynamics of peptidergic secretory granule transport are regulated by neuronal stimulation

BackgroundPeptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs) from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated. Sensory neurons also package opiate receptors in peptide-containing LDCVs, which is thought to be important in pain sensation. Since peptide granules cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides.ResultsA vector encoding soluble peptidylglycine α-hydroxylating monooxygenase fused to green fluorescent protein was constructed to address these questions in cultured primary peptidergic neurons of the trigeminal ganglion using time lapse confocal microscopy. The time course of release differs with secretagogue; the secretory response to depolarization with K+ is rapid and terminates within 15 minutes, while phorbol ester stimulation of secretion is maintained over a longer period. The data demonstrate fundamental differences between LDCV dynamics in axons and growth cones under basal conditions.ConclusionsUnder basal conditions, LDCVs move faster away from the soma than toward the soma, but fewer LDCVs travel anterograde than retrograde. Stimulation decreased average anterograde velocity and increases granule pausing. Data from antibody uptake, quantification of enzyme secretion and appearance of pHluorin fluorescence demonstrate distributed release of peptides all along the axon, not just at terminals.

Structural biology of copper trafficking.

1.1. Background The use of copper in biological systems coincides with the advent of an oxygen atmosphere about 1.7 billion years ago. The presence of O2 both allowed the oxidation of insoluble Cu(I) to the more soluble and bioavailable Cu(II) and led to the requirement for a redox active metal with potentials in the 0-800 mV range. Not only did copper meet this need, but the oxidation of Fe(II) to the insoluble Fe(III) form rendered the use of iron more energetically expensive.1-5 As a result, copper plays a key role in many proteins that react with O2. Generally, O2-reactive centers are mononuclear (type 2), dinuclear (type 3), or trinuclear (type 2 and type 3). Well studied mononuclear copper enzymes include the monooxygenases dopamine-β-hydroxylase and peptidylglycine α-hydroxylating monooxygenase as well as oxidases that also contain organic cofactors, such as amine, galactose, and lysyl oxidases.6 Dinuclear copper proteins include the O2 carrier hemocyanin and enzymes like tyrosinase and catechol oxidase.7 Copper also plays a key role in numerous electron transfer proteins. Mononuclear type 1 (blue copper) centers are found in proteins such as plastocyanin and azurin.8 The multicopper oxidases like laccase, ascorbate oxidase, and ceruloplasmin contain both a catalytic trinuclear type 2/type 3 site and an electron transfer type 1 site.9,10 The classification of copper centers into types is derived from optical and electron paramagnetic resonance (EPR) spectroscopic properties, and there are some notable exceptions, including the cysteine-bridged dinuclear CuA electron transfer site in cytochrome c oxidase11 and nitrous oxide reductase, the tetranuclear catalytic CuZ center in nitrous oxide reductase,12 and the proposed catalytic copper center in particulate methane monooxygenase.13-15 The same redox properties that render copper useful in all these metalloproteins can lead to oxidative damage in cells. Reaction of Cu(I) with hydrogen peroxide and re-reduction of Cu(II) by superoxide via Fenton and Haber-Weiss chemistry yields hydroxyl radicals that can damage proteins, lipids, and nucleic acids.16 Thus, intracellular copper concentrations must be controlled such that copper ions are provided to essential enzymes, but do not accumulate to deleterious levels. In humans, deficiencies in copper metabolism are linked to diseases such as Menkes syndrome, Wilson disease, prion diseases, and Alzheimer’s disease.17 Several classes of proteins, including membrane transporters,18-20 metallochaperones,21,22 and metalloregulatory proteins,23,24 are implicated in copper homeostasis. These proteins have two functions. First, they ensure that copper is provided to the correct proteins and cellular compartments for necessary activities. Second, these proteins detoxify excess copper. Just as copper-containing proteins and enzymes are found in all kingdoms of life, members of these groups of homeostatic proteins are also widespread,5 and have been structurally and biochemically characterized from eukaryotes and prokaryotes.

Imino-oxy Acetic Acid Dealkylation as Evidence for an Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed by Peptidylglycine α-Hydroxylating Monooxygenase

Peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl alpha-carbon in a reaction that requires O(2) and ascorbate. Subsequent dealkylation of the alpha-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a noncoupled, type II dicopper monooxygenase which activates O(2) at only a single copper atom, Cu(M). In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The (D)(V/K)(APPARENT, BIAA) decreased from 14.7 +/- 1.0 as [O(2)] --> 0 to 1.0 +/- 0.2 as [O(2)] --> infinity suggesting the dissociation rate constant from the PHM x BIAA complex decreases as [O(2)] increases; thereby, reducing the steady-state concentration of [PHM](free). BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked Cu(II)-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.

From precursor to peptide: Evidence for endomorphin biosynthesis?

Event Abstract Back to Event From precursor to peptide: Evidence for endomorphin biosynthesis? Jayapalreddy Mallareddy1, Balazs Csibrany1, Attila Keresztes1 and Géza Tóth1* 1 Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Hungary Because of their high affinity, selectivity and agonist properties, endomorphins were proposed to be the endogenous ligands of the -opioid receptors. In spite of recent advances, the biosynthetic pathway of endomorphins is still unclear. Recent findings suggested that endomorphin-2 may be produced from a human sulfate transporter protein in consecutive enzymatic reactions. Many biologically active neuropeptides and peptide hormones were shown to be synthesized from inactive glycine-extended precursors, which require a carboxy-terminal posttranslational amidation for biological activity. This reaction is catalyzed by a consecutive action of peptidylglycine-α-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL). These two enzymes are part of the peptidylglycine α-amidating monooxygenase (PAM), a bifunctional, integral membrane-bound enzyme machinery. In attempt to elicit the hypothesis that endomorphin-2 may be produced from the sulfate transporter precursor protein by the activation of the PAM enzyme system we designed and performed a series of biochemical experiments. Biological matrices, serum and brain homogenates were prepared for the in vitro metabolic studies. Endomorphin-2, Gly5-extended endomorphin-2 as well as their Dmt1-containing analogs were synthesized using SPPS methods. The biodegradation of the Gly5-extended endomorphin-2 was monitored by RP-HPLC and MALDI-TOF mass spectroscopy. Additionally, the -and -opioid receptor binding affinities of the aforementioned analogs were tested. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Developmental neurobiology and subcortical functions Citation: Mallareddy J, Csibrany B, Keresztes A and Tóth G (2009). From precursor to peptide: Evidence for endomorphin biosynthesis?. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.245 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 13 Mar 2009; Published Online: 13 Mar 2009. * Correspondence: Géza Tóth, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary, geza@brc.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jayapalreddy Mallareddy Balazs Csibrany Attila Keresztes Géza Tóth Google Jayapalreddy Mallareddy Balazs Csibrany Attila Keresztes Géza Tóth Google Scholar Jayapalreddy Mallareddy Balazs Csibrany Attila Keresztes Géza Tóth PubMed Jayapalreddy Mallareddy Balazs Csibrany Attila Keresztes Géza Tóth Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine α-hydroxylating monooxygenase (PHM)

Peptidyl α-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of α-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C 6H 5–CO–NH–CH 2–COOH) inhibit the enzyme with K i values as low as 0.5 μM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, –CO–NH–CH 2–COOH with an S-(thioacetyl)thioglycolic acid moiety, –CS–S–CH 2–COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K i values. Comparison of K i values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

Intermittent hypoxia activates peptidylglycine α-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.