Structural basis of the divergent oxygenation reactions catalyzed by the rieske nonheme iron oxygenase carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO), a Rieske nonheme iron oxygenase (RO), is a three-component system composed of a terminal oxygenase (Oxy), ferredoxin, and a ferredoxin reductase. Oxy has angular dioxygenation activity against carbazole. Previously, site-directed mutagenesis of the Oxy-encoding gene from Janthinobacterium sp.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, β-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA.

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins.

Rapid quantitative profiling of N-glycan by the glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis.

Intact-cell-based surface plasmon resonance measurements for ligand affinity evaluation of a membrane receptor.

Toward future applications to the discovery of drugs against membrane receptors on pathological cells, an intact-cell-based surface plasmon resonance (SPR) methodology has been developed. The injection of a suspension of epidermal carcinoma A431 cells (5×10(7)cells/ml), as an analyte, generated clear SPR responses to epidermal growth factor (EGF) immobilized on the sensor chip. Because the responses were competitively reduced by the free ligand EGF, added to the analyte cell suspension, they certainly reflect the specific interaction of the immobilized EGF with the extracellular region of its receptor, which is highly expressed on the surface of the A431 cells.

Quantitative evaluation of refolding conditions for a disulfide-bond-containing protein using a concise ¹⁸O-labeling technique.

A concise method was developed for quantifying native disulfide-bond formation in proteins using isotopically labeled internal standards, which were easily prepared with proteolytic ¹⁸O-labeling. As the method has much higher throughput to estimate the amounts of fragments possessing native disulfide arrangements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) than the conventional high performance liquid chromatography (HPLC) analyses, it allows many different experimental conditions to be assessed in a short time. The method was applied to refolding experiments of a recombinant neuregulin 1-β1 EGF-like motif (NRG1-β1), and the optimum conditions for preparing native NRG1-β1 were obtained by quantitative comparisons.

Verification of protein disulfide bond arrangement by in-gel tryptic digestion under entirely neutral pH conditions.

To develop a concise proteomic procedure to verify the protein disulfide bond arrangement, non-reductive trypsin digestion of neuregulin 1-beta1 (176-246), a model disulfide-containing protein, was assessed by a proteolytic (18)O-labeling analysis. As a result, the commonly used in-gel tryptic digestion method has been improved for use entirely under neutral pH conditions. With this procedure, the disulfide arrangement of proteins could represent a clinical index candidate in pathological proteomic studies.

Inhibitory effect of a dimerization-arm-mimetic peptide on EGF receptor activation.

A cyclic decapeptide was chemically synthesized that mimics the loop structure of a beta-hairpin arm of the EGF receptor, which is highly involved in receptor dimerization upon activation by ligand binding. This peptide was revealed to reduce dimer formation of the receptor in a detergent-solubilized extract of epidermoid carcinoma A431 cells and to inhibit receptor autophosphorylation at less than 10 microM in the intact cells.

Alteration of the substrate specificity of the angular dioxygenase carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized.

Fmoc-based solid phase chemical synthesis of 71-meric neuregulin 1-beta1, an epidermal growth factor-like domain.

The human neuregulin 1-beta1 (NRG1-beta1, amino acid residues 176-246) was chemically synthesized by Fmoc-based solid phase peptide synthesis (SPPS) followed by folding in a redox buffer. The biological activity of the synthesized NRG1-beta1 was confirmed by ligand-induced tyrosine phosphorylation on Chinese hamster ovary (CHO) cells expressing ErbB-4.

Crystallization and preliminary X-ray diffraction studies of the ferredoxin reductase component in the Rieske nonhaem iron oxygenase system carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component (CARDO-O) and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), catalyzes dihydroxylation at the C1 and C9a positions of carbazole. CARDO-R was crystallized at 277 K using the hanging-drop vapour-diffusion method with the precipitant PEG 8000. Two crystal types (types I and II) were obtained.

Plasmid pCAR3 contains multiple gene sets involved in the conversion of carbazole to anthranilate.

The carbazole degradative car-I gene cluster (carAaIBaIBbICIAcI) of Sphingomonas sp. strain KA1 is located on the 254-kb circular plasmid pCAR3. Carbazole conversion to anthranilate is catalyzed by carbazole 1,9a-dioxygenase (CARDO; CarAaIAcI), meta-cleavage enzyme (CarBaIBbI), and hydrolase (CarCI).

Structure of the terminal oxygenase component of angular dioxygenase, carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO) catalyzes the dihydroxylation of carbazole by angular position (C9a) carbon bonding to the imino nitrogen and its adjacent C1 carbon. This reaction is an initial degradation reaction of the carbazole degradation pathway by various bacterial strains. Only a limited number of Rieske non-heme iron oxygenase systems (ROSs) can catalyze this novel reaction, termed angular dioxygenation.

Purification and characterization of carbazole 1,9a-dioxygenase, a three-component dioxygenase system of Pseudomonas resinovorans strain CA10.

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer.

Transcriptional control of nitric oxide reductase gene (CYP55) in the fungal denitrifier Fusarium oxysporum.

Fungal denitrification is a dissimilating metabolic mechanism for nitrate and was first described in Fusarium oxysporum. Here we investigated regulatory systems of expression of CYP55, which encodes cytochrome P450 (P450nor) and is essential for the fungal denitrification. Promoter-reporter analysis of F.

Nitrate reductase-formate dehydrogenase couple involved in the fungal denitrification by Fusarium oxysporum.

Dissimilatory nitrate reductase (Nar) was solubilized and partially purified from the large particle (mitochondrial) fraction of the denitrifying fungus Fusarium oxysporum and characterized. Many lines of evidence showed that the membrane-bound Nar is distinct from the soluble, assimilatory nitrate reductase. Further, the spectral and other properties of the fungal Nar were similar to those of dissimilatory Nars of Escherichia coli and denitrifying bacteria, which are comprised of a molybdoprotein, a cytochrome b, and an iron-sulfur protein.