Posttranslational modification and heme cavity architecture of human eosinophil peroxidase-insights from first crystal structure and biochemical characterization.

Eosinophil peroxidase (EPO) is the most abundant granule protein exocytosed by eosinophils, specialized human phagocytes. Released EPO catalyzes the formation of reactive oxidants from bromide, thiocyanate, and nitrite that kill tissue-invading parasites. However, EPO also plays a deleterious role in inflammatory diseases, making it a potential pharmacological target. A major hurdle is the high similarity to the homologous myeloperoxidase (MPO), which requires a detailed understanding of the small structural differences that can be used to increase the specificity of the inhibitors. Here, we present the first crystal structure of mature leukocyte EPO at 1.6 Å resolution together with analyses of its posttranslational modifications and biochemical properties. EPO has an exceptionally high number of positively charged surface patches but only two occupied glycosylation sites. The crystal structure further revealed the existence of a light (L) and heavy (H) chain as a result of proteolytic cleavage. Detailed comparison with the structure of human MPO allows us to identify differences that may contribute to the known divergent enzymatic properties. The crystal structure revealed fully established ester links between the prosthetic group and the protein, the comparably weak imidazolate character of the proximal histidine, and the conserved structure of the catalytic amino acids and Ca2+-binding site. Prediction of the structure of unprocessed proeosinophil peroxidase allows further structural analysis of the three protease cleavage sites and the potential pro-convertase recognition site in the propeptide. Finally, EPO biosynthesis and its biochemical and biophysical properties are discussed with respect to the available data from the well-studied MPO.

Compound I Formation and Reactivity in Dimeric Chlorite Dismutase: Impact of pH and the Dynamics of the Catalytic Arginine.

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

The staphylococcal inhibitory protein SPIN binds to human myeloperoxidase with picomolar affinity but only dampens halide oxidation.

The heme enzyme myeloperoxidase (MPO) is one of the key players in the neutrophil-mediated killing of invading pathogens as part of the innate immune system. MPO generates antimicrobial oxidants, which indiscriminately and effectively kill phagocytosed pathogens. Staphylococcus aureus, however, is able to escape this fate, in part by secreting a small protein called SPIN (Staphylococcal Peroxidase Inhibitor), which specifically targets and inhibits MPO in a structurally complex manner. Here, we present the first crystal structures of the complex of SPIN-aureus and a truncated version (SPIN-truncated) with mature dimeric leukocyte MPO. We unravel the contributions of the two domains to the kinetics and thermodynamics of SPIN-aureus binding to MPO by using a broad array of complementary biochemical and biophysical methods. The C-terminal "recognition" domain is shown to mediate specific binding to MPO, while interaction of the N-terminal "inhibitory" domain is guided mainly by hydrophobic effects and thus is less sequence dependent. We found that inhibition of MPO is achieved by reducing substrate migration, but SPIN-aureus cannot completely block MPO activity. Its' effectiveness is inversely related to substrate size, with no discernible dependence on other factors. Thus, SPIN-aureus is an extremely high-affinity inhibitor and highly efficient for substrates larger than halogens. As aberrant MPO activity is implicated in a number of chronic inflammatory diseases, SPIN-aureus is the first promising protein inhibitor for specific inhibition of human MPO.

A conformation-specific ON-switch for controlling CAR T cells with an orally available drug.

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.

Identification of Activating Mutations in the Transmembrane and Extracellular Domains of EGFR.

The epidermal growth factor receptor (EGFR) is frequently mutated in human cancer, most notably non-small-cell lung cancer and glioblastoma. While many frequently occurring EGFR mutations are known to confer constitutive EGFR activation, the situation is less clear for rarely detected variants. In fact, more than 1000 distinct EGFR mutations are listed in the Catalogue of Somatic Mutations in Cancer (COSMIC), but for most of them, the functional consequence is unknown. To identify additional, previously unknown activating mutations in EGFR, we screened a randomly mutated EGFR library for constitutive EGFR phosphorylation using a recently developed high-throughput approach termed PhosphoFlowSeq. Enrichment of the well-known activating mutations S768I, T790M, and L858R validated the experimental approach. Importantly, we also identified the activating mutations S442I and L658Q located in the extracellular and transmembrane domains of EGFR, respectively. To the best of our knowledge, neither S442I nor L658Q has been associated with an activating phenotype before. However, both have been detected in cancer samples. Interestingly, molecular dynamics (MD) simulations suggest that the L658Q mutation located in the hydrophobic transmembrane region forms intermolecular hydrogen bonds, thereby promoting EGFR dimerization and activation. Based on these findings, we screened the COSMIC database for additional hydrophilic mutations in the EGFR transmembrane region and indeed detected moderate constitutive activation of EGFR-G652R. Together, this study demonstrates that unbiased screening for activating mutations in EGFR not only yields well-established substitutions located in the kinase domain but also activating mutations in other regions of EGFR, including the extracellular and transmembrane domains.

On 'Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium' by Jeffrey K. Glenn and Michael H. Gold.

This paper by Jeffrey K. Glenn and Michael H. Gold (Department of Chemical, Biological, and Environmental Sciences, Oregon Graduate Center) reported for the first time the purification and characterization of a manganese peroxidase. It was shown that the extracellular heme b-containing oxidoreductase purified from the basidiomycete Phanerochaete chrysosporium requires hydrogen peroxide and Mn(II) for the oxidation of a variety of different compounds. This discovery in 1985 was the prelude to countless research papers on structure-function relationships of manganese peroxidases, their ecological role(s) in the degradation of lignocellulose and lignin and on their relevance for industrial and commercial applications. This paper has been cited 575 times in Scopus. A Scopus search for the term manganese peroxidase yielded 6163 results (April 2022).

Reaction intermediate rotation during the decarboxylation of coproheme to heme b in C. diphtheriae.

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H2O2. Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

Arresting the Catalytic Arginine in Chlorite Dismutases: Impact on Heme Coordination, Thermal Stability, and Catalysis.

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that can decompose chlorite to chloride and molecular oxygen. They are divided in two clades that differ in oligomerization, subunit architecture, and the hydrogen-bonding network of the distal catalytic arginine, which is proposed to switch between two conformations during turnover. To understand the impact of the conformational dynamics of this basic amino acid on heme coordination, structure, and catalysis, Cld from Cyanothece sp. PCC7425 was used as a model enzyme. As typical for a clade 2 Cld, its distal arginine 127 is hydrogen-bonded to glutamine 74. The latter has been exchanged with either glutamate (Q74E) to arrest R127 in a salt bridge or valine (Q74V) that mirrors the setting in clade 1 Clds. We present the X-ray crystal structures of Q74V and Q74E and demonstrate the pH-induced changes in the environment and coordination of the heme iron by ultraviolet-visible, circular dichroism, and electron paramagnetic resonance spectroscopies as well as differential scanning calorimetry. The conformational dynamics of R127 is shown to have a significant role in heme coordination during the alkaline transition and in the thermal stability of the heme cavity, whereas its impact on the catalytic efficiency of chlorite degradation is relatively small. The findings are discussed with respect to (i) the flexible loop connecting the N-terminal and C-terminal ferredoxin-like domains, which differs in clade 1 and clade 2 Clds and carries Q74 in clade 2 proteins, and (ii) the proposed role(s) of the arginine in catalysis.

X-ray-induced photoreduction of heme metal centers rapidly induces active-site perturbations in a protein-independent manner.

Since the advent of protein crystallography, atomic-level macromolecular structures have provided a basis to understand biological function. Enzymologists use detailed structural insights on ligand coordination, interatomic distances, and positioning of catalytic amino acids to rationalize the underlying electronic reaction mechanisms. Often the proteins in question catalyze redox reactions using metal cofactors that are explicitly intertwined with their function. In these cases, the exact nature of the coordination sphere and the oxidation state of the metal is of utmost importance. Unfortunately, the redox-active nature of metal cofactors makes them especially susceptible to photoreduction, meaning that information obtained by photoreducing X-ray sources about the environment of the cofactor is the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species by X-ray radiation. We show that a dose of ∼40 kilograys already yields 50% ferrous iron in a heme protein crystal. We also demonstrate that the kinetics of photoreduction are completely independent from variables unique to the different samples tested. The photoreduction-induced structural rearrangements around the metal cofactors have to be considered when biochemical data of ferric proteins are rationalized by constraints derived from crystal structures of reduced enzymes.

Inhibition of Myeloperoxidase.

Myeloperoxidase participates in innate immune defense mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. This has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, based on the profound knowledge of structure and function of MPO and its biochemical and biophysical differences with the other homologous human peroxidases, various rational and high-throughput screening attempts were performed in developing specific irreversible and reversible inhibitors. The most prominent candidates as well as MPO inhibitors already studied in clinical trials are introduced and discussed.

In Vitro Heme Coordination of a Dye-Decolorizing Peroxidase-The Interplay of Key Amino Acids, pH, Buffer and Glycerol.

Dye-decolorizing peroxidases (DyPs) have gained interest for their ability to oxidize anthraquinone-derived dyes and lignin model compounds. Spectroscopic techniques, such as electron paramagnetic resonance and optical absorption spectroscopy, provide main tools to study how the enzymatic function is linked to the heme-pocket architecture, provided the experimental conditions are carefully chosen. Here, these techniques are used to investigate the effect of active site perturbations on the structure of ferric P-class DyP from Klebsiella pneumoniae (KpDyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A). Arg-232 is found to be important for maintaining the heme distal architecture and essential to facilitate an alkaline transition. The latter is promoted in absence of Asp-143. Furthermore, the non-innocent effect of the buffer choice and addition of the cryoprotectant glycerol is shown. However, while unavoidable or indiscriminate experimental conditions are pitfalls, careful comparison of the effects of different exogenous molecules on the electronic structure and spin state of the heme iron contains information about the inherent flexibility of the heme pocket. The interplay between structural flexibility, key amino acids, pH, temperature, buffer and glycerol during in vitro spectroscopic studies is discussed with respect to the poor peroxidase activity of bacterial P-class DyPs.

Distinct Fcα receptor N-glycans modulate the binding affinity to immunoglobulin A (IgA) antibodies.

Human immunoglobulin A (IgA) is the most prevalent antibody class at mucosal sites with an important role in mucosal defense. Little is known about the impact of N-glycan modifications of IgA1 and IgA2 on binding to the Fcα receptor (FcαRI), which is also heavily glycosylated at its extracellular domain. Here, we transiently expressed human epidermal growth factor receptor 2 (HER2)-binding monomeric IgA1, IgA2m(1), and IgA2m(2) variants in Nicotiana benthamiana ΔXT/FT plants lacking the enzymes responsible for generating nonhuman N-glycan structures. By coinfiltrating IgA with the respective glycan-modifying enzymes, we generated IgA carrying distinct homogenous N-glycans. We demonstrate that distinctly different N-glycan profiles did not influence antigen binding or the overall structure and integrity of the IgA antibodies but did affect their thermal stability. Using size-exclusion chromatography, differential scanning and isothermal titration calorimetry, surface plasmon resonance spectroscopy, and molecular modeling, we probed distinct IgA1 and IgA2 glycoforms for binding to four different FcαRI glycoforms and investigated the thermodynamics and kinetics of complex formation. Our results suggest that different N-glycans on the receptor significantly contribute to binding affinities for its cognate ligand. We also noted that full-length IgA and FcαRI form a mixture of 1:1 and 1:2 complexes tending toward a 1:1 stoichiometry due to different IgA tailpiece conformations that make it less likely that both binding sites are simultaneously occupied. In conclusion, N-glycans of human IgA do not affect its structure and integrity but its thermal stability, and FcαRI N-glycans significantly modulate binding affinity to IgA.

Reaction of human peroxidasin 1 compound I and compound II with one-electron donors.

Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M-1 s-1 (urate) to (4.8 ± 0.1) × 105 M-1 s-1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M-1 s-1 (tyrosine) to (7.7 ± 0.1) × 103 M-1 s-1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

The leucine-rich repeat domain of human peroxidasin 1 promotes binding to laminin in basement membranes.

Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.e. leucine-rich repeat domain (LRR), immunoglobulin domains, peroxidase domain, von Willebrand factor type C domain] of PXDN interact with the polymeric networks of the extracellular matrix (ECM), and how these interactions integrate and regulate the enzyme's cross-linking activity, without imparting oxidative damage to the ECM. In this study, we probed the interactions of four PXDN constructs with different domain compositions with components of a basement membrane extract by immunoprecipitation. Strong binding of the LRR-containing construct was detected with the major ECM protein laminin. Analysis of these interactions by surface plasmon resonance spectroscopy revealed similar kinetics and affinities of binding of the LRR-containing construct to human and murine laminin-111, with calculated dissociation constants of 1.0 and 1.5 µM, respectively. The findings are discussed with respect to the recently published in-solution structures of the PXDN constructs and the proposed biological role of this peroxidase.

Engineered IgG1-Fc--one fragment to bind them all.

The crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is a very attractive scaffold for the design of novel therapeutics due to its quality of uniting all essential antibody functions. This article reviews the functionalization of this homodimeric glycoprotein by diversification of structural loops of CH3 domains for the design of Fcabs, i.e. antigen-binding Fc proteins. It reports the design of libraries for the selection of nanomolar binders with wildtype-like in vivo half-life and correlation of Fc receptor binding and ADCC. The in vitro and preclinical biological activity of selected Fcabs is compared with that of clinically approved antibodies. Recently, the great potential of the scaffold for the development of therapeutics for clinical use has been shown when the HER2-binding Fcab FS102 entered clinical phase I. Furthermore, methods for the engineering of biophysical properties of Fcabs applicable to proteins in general are presented as well as the different approaches in the design of heterodimeric Fc-based scaffolds used in the generation of bispecific monoclonal antibodies. Finally, this work critically analyzes and compares the various efforts in the design of highly diverse and functional libraries that have been made in the engineering of IgG1-Fc and structurally similar scaffolds.

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage.

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide-dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 Å) and the variants D143A (1.3 Å), R232A (1.9 Å), and D143A/R232A (1.1 Å). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin π-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.

Secreted heme peroxidase from Dictyostelium discoideum: Insights into catalysis, structure, and biological role.

Oxidation of halides and thiocyanate by heme peroxidases to antimicrobial oxidants is an important cornerstone in the innate immune system of mammals. Interestingly, phylogenetic and physiological studies suggest that homologous peroxidases are already present in mycetozoan eukaryotes such as Dictyostelium discoideum This social amoeba kills bacteria via phagocytosis for nutrient acquisition at its single-cell stage and for antibacterial defense at its multicellular stages. Here, we demonstrate that peroxidase A from D. discoideum (DdPoxA) is a stable, monomeric, glycosylated, and secreted heme peroxidase with homology to mammalian peroxidases. The first crystal structure (2.5 Å resolution) of a mycetozoan peroxidase of this superfamily shows the presence of a post-translationally-modified heme with one single covalent ester bond between the 1-methyl heme substituent and Glu-236. The metalloprotein follows the halogenation cycle, whereby compound I oxidizes iodide and thiocyanate at high rates (>108 m-1 s-1) and bromide at very low rates. It is demonstrated that DdPoxA is up-regulated and likely secreted at late multicellular development stages of D. discoideum when migrating slugs differentiate into fruiting bodies that contain persistent spores on top of a cellular stalk. Expression of DdPoxA is shown to restrict bacterial contamination of fruiting bodies. Structure and function of DdPoxA are compared with evolutionary-related mammalian peroxidases in the context of non-specific immune defense.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese Peroxidase In Vitro.

Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCE Establishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.