Noboru Ohsawa

Physiological function of bromoperoxidase in the red marine alga, Corallina pilulifera: production of bromoform as an allelochemical and the simultaneous elimination of hydrogen peroxide

The physiological function of vanadium-bromoperoxidase (BPO) in the marine red alga, Corallina pilulifera, has been characterized from the viewpoint of allelochemical formation. The algae emit bromoform (CHBr3) depending on the enzyme activity level in vivo (Itoh, N., Shinya, M., 1994. Seasonal evolution of bromomethanes from coralline algae and its effect on atmospheric ozone. Marine Chemistry 45, 95-103). We demonstrated that bromoform produced by C. pilulifera played an important role in eliminating epiphytic organisms, especially microalgae on the surface. Such data suggest a strong relationship between the coralline algae and the coralline flat (deforested area in the marine environment: called isoyake in Japanese). Lithophyllum yessoense, the main inhabitant of coralline flats in Japan, produced a lower level of CHBr3 than C. pilulifera, and showed BPO activity. On the other hand, the seasonal change of BPO activity in C. pilulifera in vivo was in proportion to superoxide dismutase (SOD) activity and in inverse proportion to catalase activity. The phenomenon implies that BPO could be a potential substitute for catalase, because the enzyme catalyzes an efficient Br(-)-dependent catalase reaction.

Purification and Characterization of a Monohalomethane-producing Enzyme S-adenosyl-L-methionine: Halide Ion Methyltransferase from a Marine Microalga, Pavlova pinguis

A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The Km for iodide and SAM were 12 mM and 12 μM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I-,Br-, and Cl- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.

Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin.

The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35u2009kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15u2009kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Å resolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs.

Involvement of S -adenosylmethionine-dependent halide/thiol methyltransferase (HTMT) in methyl halide emissions from agricultural plants: isolation and characterization of an HTMT-coding gene from Raphanus sativus (daikon radish)

BackgroundBiogenic emissions of methyl halides (CH3Cl, CH3Br and CH3I) are the major source of these compounds in the atmosphere; however, there are few reports about the halide profiles and strengths of these emissions. Halide ion methyltransferase (HMT) and halide/thiol methyltransferase (HTMT) enzymes concerning these emissions have been purified and characterized from several organisms including marine algae, fungi, and higher plants; however, the correlation between emission profiles of methyl halides and the enzymatic properties of HMT/HTMT, and their role in vivo remains unclear.ResultsThirty-five higher plant species were screened, and high CH3I emissions and HMT/HTMT activities were found in higher plants belonging to the Poaceae family, including wheat (Triticum aestivum L.) and paddy rice (Oryza sativa L.), as well as the Brassicaceae family, including daikon radish (Raphanus sativus). The in vivo emission of CH3I clearly correlated with HMT/HTMT activity. The emission of CH3I from the sprouting leaves of R. sativus, T. aestivum and O. sativa grown hydroponically increased with increasing concentrations of supplied iodide. A gene encoding an S-adenosylmethionine halide/thiol methyltransferase (HTMT) was cloned from R. sativus and expressed in Escherichia coli as a soluble protein. The recombinant R. sativus HTMT (RsHTMT) was revealed to possess high specificity for iodide (I-), bisulfide ([SH]-), and thiocyanate ([SCN]-) ions.ConclusionThe present findings suggest that HMT/HTMT activity is present in several families of higher plants including Poaceae and Brassicaceae, and is involved in the formation of methyl halides. Moreover, it was found that the emission of methyl iodide from plants was affected by the iodide concentration in the cultures. The recombinant RsHTMT demonstrated enzymatic properties similar to those of Brassica oleracea HTMT, especially in terms of its high specificity for iodide, bisulfide, and thiocyanate ions. A survey of biogenic emissions of methyl halides strongly suggests that the HTM/HTMT reaction is the key to understanding the biogenesis of methyl halides and methylated sulfur compounds in nature.

Bromoperoxidase in Corallina pilulifera is regulated by its vanadate content

Abstract Seasonal changes in bromoperoxidase activity in coralline algae (Corallinaceae) are responsible for the production of volatile halogenated compounds. SDS-polyacrylamide gel electrophoresis (PAGE) of a crude protein extract showed that the concentration of this enzyme was almost constant throughout the year. Therefore, the enzyme activity in vivo changed seasonally due to a structural alteration. To elucidate this, the metal content of this enzyme at different states of activity was measured. The results revealed that the enzyme activity is controlled by the incorporation of vanadate ions, less than 1.2 mol mol enzyme −1 , in the active site of the enzyme.

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis

Abstract Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel β4 subunit explain its role in cell–cell adhesion

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary β subunits, designated as β1/β1B–β4 (encoded by SCN1B–4B, respectively), which also function in cell–cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the β4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the β4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted β4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the β4 cis dimer contributes to the trans homophilic interaction of β4 in cell–cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of β4 affects α–β4 complex formation. These observations provide the structural basis for the parallel dimer formation of β4 in VGSCs and reveal its mechanism in cell–cell adhesion.

Phosphorylated and non-phosphorylated HCK kinase domains produced by cell-free protein expression.

Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21u202fcells harboring a plasmid encoding a set of chaperone genes, dnaK, dnaJ, and grpE. We explored cell-disruption procedures and constructed an efficient protein synthesis system. Employing this system, we produced the kinase domain of human hematopoietic cell kinase (HCK) to obtain further structural information about its molecular interaction with one of its inhibitors, previously developed by our group (RK-20449). Lower reaction temperature improved the solubility, and addition of a protein phosphatase (YpoH) facilitated the homogeneous production of the non-phosphorylated kinase domain. Crystals of the purified product were obtained and the kinase-inhibitor complex structure was solved at 1.7u202fÅ resolution. In addition, results of kinase activity measurement, using a synthetic substrate, showed that the kinase activity was facilitated by autophosphorylation at Tyr416, as confirmed by the peptide mass mapping.

Structural Basis for the Inhibition of Cyclin G-Associated Kinase by Gefitinib

Abstract Gefitinib is the molecular target drug for advanced non‐small‐cell lung cancer. The primary target of gefitinib is the positive mutation of epidermal growth factor receptor, but it also inhibits cyclin G‐associated kinase (GAK). To reveal the molecular bases of GAK and gefitinib binding, structure analyses were conducted and determined two forms of the gefitinib‐bound nanobody⋅GAK kinase domain complex structures. The first form, GAK_1, has one gefitinib at the ATP binding pocket, whereas the second form, GAK_2, binds one each in the ATP binding site and a novel binding site adjacent to the activation segment C‐terminal helix, a unique element of the Numb‐associated kinase family. In the novel binding site, gefitinib binds in the hydrophobic groove around the activation segment, disrupting the conserved hydrogen bonds for the catalytic activity. These structures suggest possibilities for the development of selective GAK inhibitors for viral infections, such as the hepatitisu2005C virus.

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domains function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP–RING–ZfUBP–CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers.