Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. DATABASE: Structural data are available in the PDB database under the accession number 7ANV.

High-resolution atomic force microscopy visualization of metalloproteins and their complexes.

BACKGROUND: Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level. METHODS: In this study, the pairwise interactions of MPO, CP and LF molecules were investigated at a single-molecule level using high-resolution atomic force microscopy (AFM). Highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM) was used as a substrate for protein deposition. RESULTS: The procedure for reliable AFM investigation of metalloproteins and their complexes has been developed. Using this procedure, we have visualized, for the first time, single MPO, CP and LF molecules, characterized the morphology of MPO-CP and LF-CP complexes and confirmed the absence of direct contacts between MPO and LF molecules. Moreover, we have revealed the novel chainlike shape of MPO-CP conjugates. CONCLUSIONS: GM-HOPG was shown to be a convenient substrate for AFM investigation of metalloproteins and their complexes. Direct AFM visualization of MPO-CP and LF-CP complexes, on the one hand, complements previous data obtained from the "bulk techniques" and, on the other hand, provides new insight into the ultrastructure of MPO-CP complexes. GENERAL SIGNIFICANCE: The obtained results contribute to the better understanding of regulation of inflammation and oxidation stress mediated by collaborative action of the metalloproteins such as MPO, CP and LF.

Importance of the surface chemistry of nanoparticles on peroxidase-like activity.

We report the studies on origin of peroxidase-like activity for gold nanoparticles, as well as the impact from morphology and surface charge of nanoparticles. For this purpose, we have synthesized hollow gold nanospheres (HAuNS) and gold nanorods (AuNR) with different morphology and surface chemistry to investigate their influence on the catalytic activity. We found that citrate-capped HAuNS show catalyzing efficiency in oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide (H2O2) and it is superior to that of cetyltrimethylammonium bromide (CTAB)-capped AuNR. The kinetics of catalytic activities from HAuNS and AuNR were respectively studied under varied temperatures. The results indicated that surface chemistry rather than morphology of nanoparticles plays an important role in the catalytic reaction of substrate. Furthermore, influencing factors such as pH, amount of nanoparticle and H2O2 concentration were also investigated on HAuNS-catalyzed system. The great impact of nanoparticle surface properties on catalytic reactions makes a paradigm in constructing nanozymes as peroxidase mimic for sensing application.

An intramolecular disulfide bond designed in myoglobin fine-tunes both protein structure and peroxidase activity.

Disulfide bond plays crucial roles in stabilization of protein structure and in fine-tuning protein functions. To explore an approach for rational heme protein design, we herein rationally introduced a pair of cysteines (F46C/M55C) into the scaffold of myoglobin (Mb), mimicking those in native neuroglobin. Molecular modeling suggested that it is possible for Cys46 and Cys55 to form an intramolecular disulfide bond, which was confirmed experimentally by ESI-MS analysis, DTNB reaction and CD spectrum. Moreover, it was shown that the spontaneously formed disulfide bond of Cys46-Cys55 fine-tunes not only the heme active site structure, but also the protein functions. The substitution of Phe46 with Ser46 in F46S Mb destabilizes the protein while facilitates H2O2 activation. Remarkably, the formation of an intramolecular disulfide bond of Cys46-Cys55 in F46C/M55C Mb improves the protein stability and regulates the heme site to be more favorable for substrate binding, resulting in enhanced peroxidase activity. This study provides valuable information of structure-function relationship for heme proteins regulated by an intramolecular disulfide bond, and also suggests that construction of such a covalent bond is useful for design of functional heme proteins.

Ceruloplasmin has two nearly identical sites that bind myeloperoxidase.

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120degrees, which, in turn, exerts tension on MPO and results in conformational change.

A new antioxidant with dual functions as a peroxidase and chaperone in Pseudomonas aeruginosa.

Thiol-based peroxiredoxins (Prxs) are conserved throughout all kingdoms. We have found that a conserved typical 2-Cys Prx-like protein (PaPrx) from Pseudomonas aeruginosa bacteria displays diversity in its structure and apparent molecular weight (MW), and can act alternatively as a peroxidase and molecular chaperone. We have also identified a regulatory factor involved in this structural and functional switching. Exposure of P. aeruginosa to hydrogen peroxide (H2O2) causes PaPrx to convert from a high MW (HMW) complex to a low MW (LMW) form, which triggers a chaperone to peroxidase functional switch. This structural switching is primarily guided by either the thioredoxin (Trx) or glutathione (GSH) systems. Furthermore, comparison of our structural data [native and non-reducing polyacrylamide gel electrophoresis (PAGE) analysis, size exclusion chromatography (SEC) analysis, and electron microscopy (EM) observations] and enzymatic analyses (peroxidase and chaperone assay) revealed that the formation of oligomeric HMW complex structures increased chaperone activity of PaPrx. These results suggest that multimerization of PaPrx complexes promotes chaperone activity, and dissociation of the complexes into LMW species enhances peroxidase activity. Thus, the dual functions of PaPrx are clearly associated with their ability to form distinct protein structures.

Molecular characterization and immunohistochemical localization of palmdelphin, a cytosolic isoform of the paralemmin protein family implicated in membrane dynamics.

Palmdelphin is a newly identified cytosolic isoform of paralemmin-1, a lipid raft-associated protein implicated in cell shape control. Like paralemmin-1, palmdelphin is phosphorylated, giving rise to electrophoretic band heterogeneity that is most pronounced in the brain. In ultracentrifugation and gel filtration palmdelphin behaves as a non-globular monomer. Its C-terminal region binds glutamine synthetase. Immunohistochemical analysis of the rat brain shows a prominent localization of palmdelphin in the cerebral cortex, hippocampus, amygdala, septum, indusium griseum, piriform cortex, nucleus supraopticus, and nucleus of the lateral olfactory tract. Many of the circumscript palmdelphin-positive areas are related to the olfactory system. Immunoperoxidase electron microscopy reveals a discontinuous distribution of palmdelphin immunoreactivity, in the form of spots scattered throughout the cytoplasm of selected neuronal perikarya and dendrites, including dendritic spines, often in association with endomembranes, and in a pattern which is similar to that of the cytoplasmic fraction of paralemmin-1. In subcellular fractionation experiments palmdelphin behaves as a cytosolic protein which, however, can be partially recruited from cytosol to the detergent-resistant fraction of a membrane/cytoskeletal cell ghost preparation. These observations suggest that palmdelphin may peripherally associate with endomembranes or cytoskeleton-linked structures.

Si nanocolumns as novel nanostructured supports for enzyme immobilization.

Silicon nanocolumns have been used as novel supports for the high-density immobilization of enzymes. Silicon nanocolumns with diameters of ca. 50-100 nm and a height of 1 micron were constructed using glancing angle deposition. The surfaces were successively treated with 3-aminopropyltriethoxysilane (APTES) and then with an amine reactive polymer, poly(ethylene-alt-maleic anhydride), to attach soybean peroxidase (SBP) to the support. Optimal coverage of APTES, polymer, and SBP was obtained for incorporation of enzyme onto the sidewalls of the nanocolumns. SBP immobilized on the silicon nanocolumns demonstrated an enhancement in biocatalytic activity of 160% over that of the enzyme immobilized on flat silicon wafers with the same projected area. The enzymatic activity decreased with progressive washes for both supports. This decrease in the activity of enzyme was found to be primarily due to the intrinsic deactivation of immobilized enzyme on the silicon surface. Designing nanocolumns with optimal dimensions, spacing, and surface chemistry may lead to the development of high-density arrays of proteins for applications in biotechnology.

Effects of hyperthermal stress on the ultrastructure of platelets with reference to the localization of platelet peroxidase and fibrinogen in vivo.

Ultrastructure of platelets with the localization of platelet peroxidase and fibrinogen through 3-min 47 degrees C hot-spring bathing was investigated in eight healthy volunteers. The mean sublingual temperature rose about 1.8 degrees C 5 min after the start of bathing. The frequencies of fold, pseudopods, vacuoles, and centralization were increased after bathing. Platelet peroxidase activity was decreased after bathing. Furthermore, fibrinogen was decreased in alpha-granules after bathing. Thus, hyperthermal stress in vivo may activate platelets, resulting in consumption of platelet peroxidase and fibrinogen.