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.

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 sp.

Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III: Propionate interactions and porphyrin core deformation.

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research.

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.

Impact of the dynamics of the catalytic arginine on nitrite and chlorite binding by dimeric chlorite dismutase.

Chlorite dismutases (Clds) are heme b containing oxidoreductases able to decompose chlorite to chloride and molecular oxygen. This work analyses the impact of the distal, flexible and catalytic arginine on the binding of anionic angulate ligands like nitrite and the substrate chlorite. Dimeric Cld from Cyanothece sp.

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 (KDyP) and three variants of the main distal residues (D143A, R232A and D143A/R232A).

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.

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

Chlorite dismutases (Clds) are heme -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 sp.

Understanding molecular enzymology of porphyrin-binding α + β barrel proteins - One fold, multiple functions.

There is a high functional diversity within the structural superfamily of porphyrin-binding dimeric α + β barrel proteins. In this review we aim to analyze structural constraints of chlorite dismutases, dye-decolorizing peroxidases and coproheme decarboxylases in detail. We identify regions of structural variations within the highly conserved fold, which are most likely crucial for functional specificities.

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.

Actinobacterial Coproheme Decarboxylases Use Histidine as a Distal Base to Promote Compound I Formation.

Coproheme decarboxylases (ChdCs) catalyze the final step in heme biosynthesis of monoderm and some diderm bacteria. In this reaction, coproheme is converted to heme via monovinyl monopropionate deuteroheme (MMD) in two consecutive decarboxylation steps. In Firmicutes decarboxylation of propionates 2 and 4 of coproheme depend on hydrogen peroxide and the presence of a catalytic tyrosine.

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.

Redox Cofactor Rotates during Its Stepwise Decarboxylation: Molecular Mechanism of Conversion of Coproheme to Heme .

Coproheme decarboxylase (ChdC) catalyzes the last step in the heme biosynthesis pathway of monoderm bacteria with coproheme acting both as redox cofactor and substrate. Hydrogen peroxide mediates the stepwise decarboxylation of propionates 2 and 4 of coproheme. Here we present the crystal structures of coproheme-loaded ChdC from (LmChdC) and the three-propionate intermediate, for which the propionate at position 2 (p2) has been converted to a vinyl group and is rotated by 90° compared to the coproheme complex structure.

Redox thermodynamics of B-class dye-decolorizing peroxidases.

With >5000 annotated genes dye-decolorizing peroxidases (DyPs) represent a heme b peroxidase family of broad functional diversity. Bacterial B-class DyPs are poor peroxidases of unknown physiological function. Hydrogen peroxide efficiently mediates the rapid formation of Compound I in B-class DyPs, which, however, is stable and shows modest reactivity towards organic and inorganic electron donors.

Monomeric and homotrimeric solution structures of truncated human peroxidasin 1 variants.

Human peroxidasin 1 is a multidomain peroxidase situated in the basement membrane. The iron enzyme with covalently bound heme oxidizes bromide to hypobromous acid which facilitates the formation of distinct sulfilimine cross-links in the collagen IV network and therefore contributes to its mechanical stability. Additional to the catalytically active peroxidase domain peroxidasin comprises a leucine rich repeat domain, four Ig domains and a C-terminal von Willebrand factor type C module (VWC).

The hydrogen bonding network of coproheme in coproheme decarboxylase from Listeria monocytogenes: Effect on structure and catalysis.

Coproheme decarboxylase (ChdC) catalyzes the oxidative decarboxylation of coproheme to heme b, i.e. the last step in the recently described coproporphyrin-dependent pathway.

A constitutive active allele of the transcription factor Msn2 mimicking low PKA activity dictates metabolic remodeling in yeast.

In yeast, protein kinase A (PKA) adjusts transcriptional profiles, metabolic rates, and cell growth in accord with carbon source availability. PKA affects gene expression mostly via the transcription factors Msn2 and Msn4, two key regulators of the environmental stress response. Here we analyze the role of the PKA-Msn2 signaling module using an Msn2 allele that harbors serine-to-alanine substitutions at six functionally important PKA motifs (Msn2A6) .

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 (DyP) (1.

Posttranslational modification of heme in peroxidases - Impact on structure and catalysis.

Four heme peroxidase superfamilies arose independently in evolution. Only in the peroxidase-cyclooxygenase superfamily the prosthetic group is posttranslationally modified (PTM). As a consequence these peroxidases can form one, two or three covalent bonds between heme substituents and the protein.

Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e.

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT).