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. strain J3 generated the I262V, F275W, Q282N, and Q282Y Oxy derivatives, which showed oxygenation capabilities different from those of the wild-type enzyme. To understand the structural features resulting in the different oxidation reactions, we determined the crystal structures of the derivatives, both free and complexed with substrates. The I262V, F275W, and Q282Y derivatives catalyze the lateral dioxygenation of carbazole with higher yields than the wild type. A previous study determined the crystal structure of Oxy complexed with carbazole and revealed that the carbonyl oxygen of Gly178 hydrogen bonds with the imino nitrogen of carbazole. In these derivatives, the carbazole was rotated approximately 15, 25, and 25°, respectively, compared to the wild type, creating space for a water molecule, which hydrogen bonds with the carbonyl oxygen of Gly178 and the imino nitrogen of carbazole. In the crystal structure of the F275W derivative complexed with fluorene, C-9 of fluorene, which corresponds to the imino nitrogen of carbazole, was oriented close to the mutated residue Trp275, which is on the opposite side of the binding pocket from the carbonyl oxygen of Gly178. Our structural analyses demonstrate that the fine-tuning of hydrophobic residues on the surface of the substrate-binding pocket in ROs causes a slight shift in the substrate-binding position that, in turn, favors specific oxygenation reactions toward various substrates.

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, ß-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. Three peptides with Kd values between 8-147 µM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.

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. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.

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. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.

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.

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. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.

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. The type I crystal diffracted to a maximum resolution of 2.80 A and belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 158.7, c = 81.4 A. The type II crystal was obtained in drops from which type I crystals had been removed; it diffracted to 2.60 A resolution and belonged to the same space group, with unit-cell parameters a = b = 161.8, c = 79.5 A.

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. Angular dioxygenation is also involved in the degradation pathways of carbazole-related compounds, dioxin, and CARDO can catalyze the angular dioxygenation for dioxin. CARDO consists of a terminal oxygenase component (CARDO-O), and the electron transport components, ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R). CARDO-O has a homotrimeric structure, and governs the substrate specificity of CARDO. Here, we have determined the crystal structure of CARDO-O of Janthinobacterium sp. strain J3 at a resolution of 1.95A. The alpha3 trimeric overall structure of the CARDO-O molecule roughly corresponds to the alpha3 partial structures of other terminal oxygenase components of ROSs that have the alpha3beta3 configuration. The CARDO-O structure is a first example of the terminal oxygenase components of ROSs that have the alpha3 configuration, and revealed the presence of the specific loops that interact with a neighboring subunit, which is proposed to be indispensable for stable alpha3 interactions without structural beta subunits. The shape of the substrate-binding pocket of CARDO-O is markedly different from those of other oxygenase components involved in naphthalene and biphenyl degradation pathways. Docking simulations suggested that carbazole binds to the substrate-binding pocket in a manner suitable for catalysis of angular dioxygenation.

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. Formate-nitrate oxidoreductase activity was also detected in the mitochondrial fraction, which was shown to arise from the coupling of formate dehydrogenase (Fdh), Nar, and a ubiquinone/ubiquinol pool. This is the first report of the occurrence in a eukaryote of Fdh that is associated with the respiratory chain. The coupling with Fdh showed that the fungal Nar system is more similar to that involved in the nitrate respiration by Escherichia coli than that in the bacterial denitrifying system. Analyses of the mutant species of F. oxysporum that were defective in Nar and/or assimilatory nitrate reductase conclusively showed that Nar is essential for the fungal denitrification.

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-ß1 EGF-like motif (NRG1-ß1), and the optimum conditions for preparing native NRG1-ß1 were obtained by quantitative comparisons. Protein disulfide isomerase (PDI) was most effective at the reduced/oxidized glutathione ratio of 2:1 for refolding the denatured sample NRG1-ß1 with the native disulfide bonds.

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.

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). CARDO is a three-component dioxygenase, and CarAaI and CarAcI are its terminal oxygenase and ferredoxin components. The car-I gene cluster lacks the gene encoding the ferredoxin reductase component of CARDO. In the present study, based on the draft sequence of pCAR3, we found multiple carbazole degradation genes dispersed in four loci on pCAR3, including a second copy of the car gene cluster (carAaIIBaIIBbIICIIAcII) and the ferredoxin/reductase genes fdxI-fdrI and fdrII. Biotransformation experiments showed that FdrI (or FdrII) could drive the electron transfer chain from NAD(P)H to CarAaI (or CarAaII) with the aid of ferredoxin (CarAcI, CarAcII, or FdxI). Because this electron transfer chain showed phylogenetic relatedness to that consisting of putidaredoxin and putidaredoxin reductase of the P450cam monooxygenase system of Pseudomonas putida, CARDO systems of KA1 can be classified in the class IIA Rieske non-heme iron oxygenase system. Reverse transcription-PCR (RT-PCR) and quantitative RT-PCR analyses revealed that two car gene clusters constituted operons, and their expression was induced when KA1 was exposed to carbazole, although the fdxI-fdrI and fdrII genes were expressed constitutively. Both terminal oxygenases of KA1 showed roughly the same substrate specificity as that from the well-characterized carbazole degrader Pseudomonas resinovorans CA10, although slight differences were observed.

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. oxysporum CYP55 using Escherichia coli beta-galactosidase showed that the region between nucleotides -526 and -515 was critical for induction by nitrate. It contained a nucleotide sequence similar to the binding consensus sequence of the pathway-specific transcriptional factor NirA, which induces expression of the nitrate-assimilatory genes of Aspergillus nidulans in the presence of nitrate. This indicates that expression of the nitrate dissimilatory gene (CYP55) is concomitantly regulated with the nitrate-assimilatory genes. The deletion studies also indicated that the nucleotide sequence between -118 and -107, which was similar to the binding consensus of the yeast Rox1p, which represses the anoxic genes under aerobic conditions, was responsible for repression of CYP55 under aerobic conditions. These results indicate that the fungus adapts to the denitrifying conditions by a combination of NirA- and Rox1-like transcription factors.

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. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the k(cat)/K(m) for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.