Peptidylglycine Α-hydroxylating Monooxygenase Research Articles

Efficient production of peptidylglycine α-hydroxylating monooxygenase in yeast for protein C-terminal functionalization

Peptidylglycine α-hydroxylating monooxygenase (PHM) is pivotal for C-terminal amidation of bioactive peptides in animals, offering substantial potential for customized protein synthesis. However, efficient PHM production has been hindered by the complexity of animal cell culture and the absence of glycosylation in bacterial hosts. Here, we demonstrate the recombinant expression of Caenorhabditis elegans PHM in the yeast Pichia pastoris, achieving a remarkable space-time yield of 28.8 U/L/day. This breakthrough surpasses prior PHM production rates and eliminates the need for specialized cultivation equipment or complex transfection steps. Mass spectrometry revealed N-glycosylation at residue N182 of recombinant CePHM, which impacts the enzyme's activity as indicated by biochemical experiments. To showcase the utility of CePHM, we performed C-terminal amidation on ubiquitin at a substrate loading of 30 g/L, a concentration meeting the requirements for pharmaceutical peptide production. Overall, this work establishes an efficient PHM production method, promising advancements in scalable manufacturing of C-terminally modified bioactive peptides and probe proteins.

In vivo characterization of the maturation steps of a pigment dispersing factor neuropeptide precursor in the Drosophila circadian pacemaker neurons.

Pigment dispersing factor (PDF) is a key signaling molecule coordinating the neuronal network associated with the circadian rhythms in Drosophila. The precursor (proPDF) of the mature PDF (mPDF) consists of 2 motifs, a larger PDF-associated peptide (PAP) and PDF. Through cleavage and amidation, the proPDF is predicted to produce cleaved-PAP (cPAP) and mPDF. To delve into the in vivo mechanisms underlying proPDF maturation, we generated various mutations that eliminate putative processing sites and then analyzed the effect of each mutation on the production of cPAP and mPDF by 4 different antibodies in both ectopic and endogenous conditions. We also assessed the knockdown effects of processing enzymes on the proPDF maturation. At the functional level, circadian phenotypes were measured for all mutants and knockdown lines. As results, we confirm the roles of key enzymes and their target residues: Amontillado (Amon) for the cleavage at the consensus dibasic KR site, Silver (Svr) for the removal of C-terminal basic residues from the intermediates, PAP-KR and PDF-GK, derived from proPDF, and PHM (peptidylglycine-α-hydroxylating monooxygenase) for the amidation of PDF. Our results suggest that the C-terminal amidation occurs independently of proPDF cleavage. Moreover, the PAP domain is important for the proPDF trafficking into the secretory vesicles and a close association between cPAP and mPDF following cleavage seems required for their stability within the vesicles. These studies highlight the biological significance of individual processing steps and the roles of the PAP for the stability and function of mPDF which is essential for the circadian clockworks.

Front Cover: Thioether‐Containing Copper Complexes as PHM, DβM, and TβM Model Systems (Eur. J. Inorg. Chem. 1/2022)

The Front Cover shows the synergic action of copper and sulfur in thioether-containing copper monooxygenases that generate mammalian and invertebrate neurotransmitters. The review reports efforts of synthetic chemists to mimic the active sites of peptidylglycine α-hydroxylating monooxygenase, dopamine β-monooxygenase, and tyramine β-monooxygenase. N- and S-donors, the latter as thioethers, reproduce the properties of the monocopper sites that hydroxylate substrates to produce neurotransmitters. Redox properties are modulated by the thioethers to generate cupric-superoxo intermediates, perhaps acting as hemilabile donors. The cover was designed by Erika Osorio. More information can be found in the Minireview by V. Gómez-Vidales and I. Castillo.

Thioether‐Containing Copper Complexes as PHM, DβM, and TβM Model Systems

AbstractThis minireview presents efforts of synthetic chemists to mimic the active site of the copper monooxygenase enzymes peptidylglycine α‐hydroxylating monooxygenase (PHM), dopamine β‐monooxygenase (DβM), and tyramine β‐monooxygenase (TβM), all of which feature copper‐thioether ligation in their resting states. These monooxygenases are involved in the initial activation of O2 to generate neurotransmitters by the selective activation of C−H bonds of their corresponding substrates, resulting in subsequent hydroxylation. Attempts to emulate the N2S donor set provided by the two histidines and the methionine residues that coordinate to copper in the active site employ N2S and N3S‐type ligands, with the latter ones providing the best conditions for testing the reactivity of their CuI complexes with dioxygen. Identification of CuII‐superoxos as capable agents for C−H activation has provided support for these species as the key reactive intermediates in the enzymatic systems. Synthetic end‐on [CuII‐η1‐O2.−] and side‐on [CuII‐η2‐O2.−] cupric superoxos are capable of such transformations, which is related to the open question of whether the methionine S‐donor may act as a hemilabile ligand in biological systems.

Mutation to second sphere residue in peptidylglycine α-hydroxylating monooxygenase (PHM) reveals role of hydrogen-bonding network

Mutation to second sphere residue in peptidylglycine α-hydroxylating monooxygenase (PHM) reveals role of hydrogen-bonding network

Venom characterization of the bark scorpion Centruroides edwardsii (Gervais 1843): Composition, biochemical activities and in vivo toxicity for potential prey

In this study, we characterize the venom of Centruroides edwardsii, one of the most abundant scorpions in urban and rural areas of Costa Rica, in terms of its biochemical constituents and their biological activities. C. edwardsii venom is rich in peptides but also contains some higher molecular weight protein components. No phospholipase A2, hemolytic or fibrinogenolytic activities were found, but the presence of proteolytic and hyaluronidase enzymes was evidenced by zymography.Venom proteomic analysis indicates the presence of a hyaluronidase, several cysteine-rich secretory proteins, metalloproteinases and a peptidylglycine α-hydroxylating monooxygenase like-enzyme. It also includes peptides similar to the K+-channel blocker margatoxin, a dominant toxin in the venom of the related scorpion C. margaritatus. MS and N-terminal sequencing analysis also reveals the presence of Na+-channel-modulating peptides with sequence similarity to orthologs present in other scorpion species of the genera Centruroides and Tityus. We purified the hyaluronidase (which co-eluted with an allergen 5-like CRiSP) and sequenced ~60% of this enzyme. We also sequenced some venom gland transcripts that include other cysteine-containing peptides and a Non-Disulfide Bridged Peptide (NDBP).Our in vivo experiments characterizing the effects on potential predators and prey show that C. edwardsii venom induces paralysis in several species of arthropods and geckos; crickets being the most sensitive and cockroaches and scorpions the most resistant organisms tested. Envenomation signs were also observed in mice, but no lethality was reached by intraperitoneal administration of this venom up to 120 μg/g body weight.

The Dual α-Amidation System in Scorpion Venom Glands.

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.

On the Metal Cooperativity in a Dinuclear Copper-Guanidine Complex for Aliphatic C-H Bond Cleavage by Dioxygen.

Selective oxidation reactions of organic compounds with dioxygen using molecular copper complexes are of relevance to synthetic chemistry as well as enzymatic reactivity. In the enzyme peptidylglycine α-hydroxylating monooxygenase (PHM), the hydroxylating activity towards aliphatic substrates arises from the cooperative effect between two copper atoms, but the detailed mechanism has yet to be fully clarified. Herein, we report on a model complex showing hydroxylation of an aliphatic ligand initiated by dioxygen. According to DFT calculations, the proton-coupled electron-transfer (PCET) process leading to ligand hydroxylation in this complex benefits from cooperative effects between the two copper atoms. While one copper atom is responsible for dioxygen binding and activation, the other stabilizes the product of intramolecular PCET by copper-ligand charge transfer. The results of this work might pave the way for the directed utilization of cooperative effects in oxidation reactions.

Effects of copper occupancy on the conformational landscape of peptidylglycine \u03b1-hydroxylating monooxygenase

The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.

Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

Nitrite Reductase Activity in Engineered Azurin Variants.

Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.

O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking

Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment.

The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution.

The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson's disease, schizophrenia, Alzheimer's disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.

60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C.

A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM in Chlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.

Oxygen-18 Kinetic Isotope Effects of Nonheme Iron Enzymes HEPD and MPnS Support Iron(III) Superoxide as the Hydrogen Abstraction Species.

Nonheme iron oxygenases that carry out four-electron oxidations of substrate have been proposed to employ iron(III) superoxide species to initiate this reaction [Paria, S.; Que, L.; Paine, T. K. Angew. Chem. Int. Ed.2011, 50, 11129]. Here we report experimental evidence in support of this proposal. 18O KIEs were measured for two recently discovered mononuclear nonheme iron oxygenases: hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS). Competitive 18O KIEs measured with deuterated substrates are larger than those measured with unlabeled substrates, which indicates that C–H cleavage must occur before an irreversible reductive step at molecular oxygen. A similar observation was previously used to implicate copper(II) superoxide in the H-abstraction reactions catalyzed by dopamine β-monooxygenase [Tian, G. C.; Klinman, J. P. J. Am. Chem. Soc.1993, 115, 8891] and peptidylglycine α-hydroxylating monooxygenase [Francisco, W. A.; Blackburn, N. J.; Klinman, J. P. Biochemistry2003, 42, 1813].

Developing mononuclear copper-active-oxygen complexes relevant to reactive intermediates of biological oxidation reactions.

Active-oxygen species generated on a copper complex play vital roles in several biological and chemical oxidation reactions. Recent attention has been focused on the reactive intermediates generated at the mononuclear copper active sites of copper monooxygenases such as dopamine β-monooxygenase (DβM), tyramine β-monooxygenase (TβM), peptidylglycine-α-hydroxylating monooxygenase (PHM), and polysaccharide monooxygenases (PMO). In a simple model system, reaction of O2 and a reduced copper(I) complex affords a mononuclear copper(II)-superoxide complex or a copper(III)-peroxide complex, and subsequent H(•) or e(-)/H(+) transfer, which gives a copper(II)-hydroperoxide complex. A more reactive species such as a copper(II)-oxyl radical type species could be generated via O-O bond cleavage of the peroxide complex. However, little had been explored about the chemical properties and reactivity of the mononuclear copper-active-oxygen complexes due to the lack of appropriate model compounds. Thus, a great deal of effort has recently been made to develop efficient ligands that can stabilize such reactive active-oxygen complexes in synthetic modeling studies. In this Account, I describe our recent achievements of the development of a mononuclear copper(II)-(end-on)superoxide complex using a simple tridentate ligand consisting of an eight-membered cyclic diamine with a pyridylethyl donor group. The superoxide complex exhibits a similar structure (four-coordinate tetrahedral geometry) and reactivity (aliphatic hydroxylation) to those of a proposed reactive intermediate of copper monooxygenases. Systematic studies based on the crystal structures of copper(I) and copper(II) complexes of the related tridentate supporting ligands have indicated that the rigid eight-membered cyclic diamine framework is crucial for controlling the geometry and the redox potential, which are prerequisites for the generation of such a unique mononuclear copper(II)-(end-on)superoxide complex. Reactivity of a mononuclear copper(II)-alkylperoxide complex has also been examined to get insights into the intrinsic reactivity of copper(II)-peroxide species, which is usually considered as a sluggish oxidant or just a precursor of copper-oxyl radical type reactive species. However, our studies have unambiguously demonstrated that copper(II)-alkylperoxide complex can be a direct oxidant for C-H bond activation of organic substrates, when the C-H bond activation is coupled with O-O bond cleavage (concerted mechanism). The reactivity studies of these mononuclear copper(II) active-oxygen species (superoxide and alkylperoxide) will provide significantly important insights into the catalytic mechanism of copper monooxygenases as well as copper-catalyzed oxidation reactions in synthetic organic chemistry.

Synthesis and Characterization of a Dinuclear Copper Complex Bearing a Hydrophobic Cavity as a Model for Copper‐Containing Monooxygenases

AbstractThe synthesis and characterization of a dinuclear copper complex as a model of mononuclear copper‐containing monooxygenases [dopamine β‐monooxygenase (DβM), tyramine β‐monooxygenase (TβM), and peptidylglycine α‐hydroxylating monooxygenase (PHM)] are described. This model is based on a cyclophane moiety bearing two bis(picolylamine) ligands for the complexation of two copper atoms and exhibits macroscopic features equivalent to those of the enzymes: (1) the two copper centers are 11.5 Å apart (11 Å for the enzymes) and (2) there is a hydrophobic pocket consisting of a cyclophane moiety in which substrates can bind near the two copper centers.

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.

Copper coordinated ligand thioether-S and NO2(-) oxidation: relevance to the CuM site of hydroxylases.

In order to gain insight into the coordination site and oxidative activity of the CuM site of hydroxylases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM), we have synthesized, characterized and studied the oxidation chemistry of copper complexes chelated by tridentate N2Sthioether, N2Osulfoxide or N2Osulfone donor sets. The ligands are those of N-2-methylthiophenyl-2'-pyridinecarboxamide (HL1), and the oxidized variants, N-2-methylsulfenatophenyl-2'-pyridinecarboxamide (HL1(SO)), and N-2-methylsulfinatophenyl-2'-pyridinecarboxamide (HL1(SO2)). Our studies afforded the complexes [(L1)Cu(II)(H2O)](ClO4)·H2O (1·H2O), {[(L1(SO))Cu(II)(CH3CN)](ClO4)}n (2), [(L1)Cu(II)(ONO)] (3), [(L1(SO))Cu(II)(ONO)]n (4), [(L1)Cu(II)(NO3)]n (5), [(L1(SO))Cu(II)(NO3)]n (6) and [(L1(SO2))Cu(II)(NO3)] (7). Complexes 1 and 3 were described in a previous publication (Inorg. Chem., 2013, 52, 11084). The X-ray crystal structures revealed either distorted octahedral (in 2, 4-6) or square-pyramidal (in 1, 3) coordination geometry around Cu(II) ions of the complexes. In the presence of H2O2, conversion of 1→2, 3-5→6 and 6→7 occurs quantitatively via oxidation of thioether-S and/or Cu(ii) coordinated NO2(-) ions. Thioether-S oxidation of L1 also occurs when [L1](-) is reacted with [Cu(I)(CH3CN)4](ClO4) in DMF under O2, albeit low in yield (20%). Oxidations of thioether-S and NO2(-) were monitored by UV-Vis spectroscopy. Recovery of the sulfur oxidized ligands from their metal complexes allowed for their characterization by elemental analysis, (1)H NMR, FTIR and mass spectrometry.

Genetic determinants of amidating enzyme activity and its relationship with metal cofactors in human serum.

Backgroundα-amidation is a final, essential step in the biosynthesis of about half of all peptide hormones and neurotransmitters. Peptidylglycine α-amidating monooxygenase (PAM), with enzymatic domains that utilize Cu and Zn, is the only enzyme that catalyzes this reaction. PAM activity is detected in serum, but its significance and utility as a clinical biomarker remain unexplored.MethodsWe used well-established enzymatic assays specific for the peptidylglycine-α -hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domains of PAM to quantify amidating activity in the sera of 144 elderly men. Relationships between PHM and PAL activity and serum levels of their respective active-site metals, Cu and Zn, were analyzed. Study participants were also genotyped for eight non-coding single nucleotide polymorphisms (SNPs) in PAM, and relationships between genotype and serum enzyme activity and metal levels were analyzed.ResultsSerum PHM and PAL activities were normally distributed and correlated linearly with each other. Serum PAL activity, but not serum PHM activity, correlated with serum Cu; neither activity correlated with serum Zn. Study subjects possessing the minor alleles for rs32680 had lower PHM and PAL activities, and subjects with minor alleles for rs11952361 and rs10515341 had lower PHM activities.ConclusionsOur results characterize large variation in serum amidating activity and provide unique insight into its potential origin and determinants. Common non-coding polymorphisms affect serum amidating activity and Cu levels. Serum amidating activity should be explored as a biomarker for functionality in the elderly and in additional study groups.