Photocontrol of mitotic kinesin Eg5 facilitated by thiol-reactive photochromic molecules incorporated into the loop L5 functional loop
Kinesin Eg5 is a plus-end-directed microtubule-based motor that is essential for bipolar spindle formation during eukaryotic cell division. Loop L5 of mitotic kinesin Eg5 is a key region determining ATPase activity and motor function. Photochromic molecules undergo reversible isomerization in response to ultraviolet and visible light irradiation. We introduced three kinds of photochromic molecules, 4-phenylazomaleinanil (PAM), 4-(N-(2-iodoacetyl)amino)-4'-(N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (IATAB) and 3,3-dimethyl-1-(2-(2-iodoacetoxy)ethyl)-3H-1,2-dihydroindole-2-spiro-2'-(2H)-6'-nitrochromene (IASP) i...
Source: Journal of Biochemistry - March 2, 2014 Category: Biochemistry Authors: Ishikawa, K., Tamura, Y., Maruta, S. Tags: Regular Papers Source Type: research
Catalytic domain surface residues mediating catecholamine inhibition in tyrosine hydroxylase
Tyrosine hydroxylase (TH) performs the rate-limiting step in catecholamine (CA) synthesis and is a tetramer composed of regulatory, catalytic and tetramerization domains. CAs inhibit TH by binding two sites in the active site; one with high affinity and one with low affinity. Only high affinity CA binding requires the regulatory domain, believed to interact with the catalytic domain in the presence of CA. Without a crystal structure of the regulatory domain, the specific areas involved in this process are largely undefined. It is not clear whether the regulatory domain–catalytic domain interaction is asymmetrical acr...
Source: Journal of Biochemistry - March 2, 2014 Category: Biochemistry Authors: Briggs, G. D., Bulley, J., Dickson, P. W. Tags: Regular Papers Source Type: research
Structure and properties of the C-terminal {beta}-helical domain of VgrG protein from Escherichia coli O157
The bacterial Type 6 secretion system (T6SS) translocates protein toxins (also called effectors) from the cytosol of a T6SS-carrying cell to a target cell by a syringe-like supramolecular complex resembling a contractile tail of bacteriophages. Valine-glycine repeat protein G (VgrG) proteins, which are the homologues of the gp27-gp5 (gene product) cell puncturing complex of bacteriophage T4, are considered to be located at the attacking tip of the bacterial T6SS apparatus. Here, we over-expressed six VgrG proteins from pathogenic Escherichia coli O157 and CFT073 strains. Purified VgrG1 of E. coli O157 and c3393 of E. coli ...
Source: Journal of Biochemistry - March 2, 2014 Category: Biochemistry Authors: Uchida, K., Leiman, P. G., Arisaka, F., Kanamaru, S. Tags: Regular Papers Source Type: research
The N-terminus and Tudor domains of Sgf29 are important for its heterochromatin boundary formation function
In this study, we describe the characterization of one of these boundary genes, named SGF29, which was previously reported as a component of the SAGA, SLIK, ADA and HAT-A2 complex. A domain analysis of Sgf29 identified two minimal regions that can function as individual boundaries. The N-terminal minimal region comprising amino acids 1–12, which has not been defined as a functional domain, showed stronger boundary formation ability than the C-terminal minimal region comprising amino acids 110–255, which contains Tudor domains. Together with Ada2, Ada3 and Sgf29, which are all components of SAGA, Gcn5 acetylates...
Source: Journal of Biochemistry - March 2, 2014 Category: Biochemistry Authors: Kamata, K., Goswami, G., Kashio, S., Urano, T., Nakagawa, R., Uchida, H., Oki, M. Tags: Regular Papers Source Type: research
AMPK-sensitive cellular transport
The energy sensing AMP-activated protein kinase (AMPK) regulates cellular and whole-body energy balance through stimulating catabolic ATP-generating and suppressing anabolic ATP-consuming pathways thereby helping cells survive during energy depletion. The kinase has previously been reported to be either directly or indirectly involved in the regulation of several carriers, channels and pumps of high significance in cellular physiology. Thus AMPK provides a necessary link between cellular energy metabolism and cellular transport activity. Better understanding of the AMPK role in cellular transport offers a potential for imp...
Source: Journal of Biochemistry - March 2, 2014 Category: Biochemistry Authors: Dermaku-Sopjani, M., Abazi, S., Faggio, C., Kolgeci, J., Sopjani, M. Tags: JB Reviews Source Type: research
Multifaceted roles of Furry proteins in invertebrates and vertebrates
Furry (Fry) is a large protein that is evolutionarily conserved from yeast to human. Fry and its orthologues in invertebrates (termed Tao3p in budding yeast, Mor2p in fission yeast, Sax-2 in nematode and Fry in fruit fly) genetically and physically interact with nuclear Dbf2-related (NDR) kinases (termed Cbk1p in budding yeast, Orb6p in fission yeast, Sax-1 in nematode and Trc in fruitfly), and function as activators or scaffolds of these kinases. Fry-NDR kinase signals are implicated in the control of polarized cell growth and morphogenesis in yeast, neurite outgrowth in nematode, and epidermal morphogenesis and dendritic...
Source: Journal of Biochemistry - March 2, 2014 Category: Biochemistry Authors: Nagai, T., Mizuno, K. Tags: JB Reviews Source Type: research
Structural insights into the function of a thermostable copper-containing nitrite reductase
Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO–2) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate-bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique 1-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mo...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Fukuda, Y., Tse, K. M., Lintuluoto, M., Fukunishi, Y., Mizohata, E., Matsumura, H., Takami, H., Nojiri, M., Inoue, T. Tags: Regular Papers Source Type: research
Solution structure of the chitin-binding domain 1 (ChBD1) of a hyperthermophilic chitinase from Pyrococcus furiosus
A chitinase, from Pyrococcus furiosus, is a hyperthermophilic glycosidase that effectively hydrolyses both α and β crystalline chitin. This chitinase has unique structural features; it contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBD1 and ChBD2). We have determined the structure of ChBD1, which significantly enhances the activity of the catalytic domains, by nuclear magnetic resonance spectroscopy. The overall structure of ChBD1 had a compact and globular architecture consisting of three anti-parallel β-strands, similar to those of other proteins classified into carbohydrate...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Mine, S., Nakamura, T., Sato, T., Ikegami, T., Uegaki, K. Tags: Regular Papers Source Type: research
Arabidopsis thaliana mitochondrial EF-G1 functions in two different translation steps
In this study, we phylogenetically and biochemically characterized Arabidopsis thaliana EF-G1mt (AtEF-G1mt) to clarify the factor responsible for the dissociation of ribosomes in plant mitochondria. We showed that eukaryotic EF-G1mts form one monophyletic group separated from bacterial EF-G and are classified into five sister groups. AtEF-G1mt is classified into a different group from its human counterpart. We also demonstrated that AtEF-G1mt catalyses both translocation and ribosomal dissociation, unlike in humans. Meanwhile, AtEF-G1mt is resistant to fusidic acid, an inhibitor of bacterial EF-G. Here, we propose that the...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Suematsu, T., Watanabe, O., Kita, K., Yokobori, S.-i., Watanabe, Y.-i. Tags: Regular Papers Source Type: research
Regulation of eukaryotic-like protein kinase activity of DspA from Myxococcus xanthus by autophosphorylation
A Myxococcus xanthus DspA contains 12 subdomains characteristic of eukaryotic-like protein kinases but with an atypical sequence, RDxSPHN, in the catalytic loop, different from the consensus motifs observed in Ser/Thr kinases (RDxKxxN) or Tyr kinases (RDx(A/R)A(A/R)N). DspA phosphorylated myelin basic protein (MBP) on Ser and Thr residues. Mutations of the SPHN motif within the catalytic loop to KPHN or KPEN for Ser/Thr kinases, AARN for Tyr kinases and TPHN or TSHN for Dictyostelium Tyr kinases markedly reduced autophosphorylation and kinase activities. Phosphorylation assays, Western blot analysis and mutational analysis...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Okamoto, R., Takegawa, K., Kimura, Y. Tags: Regular Papers Source Type: research
Identification of a novel flavonoid glycoside sulfotransferase in Arabidopsis thaliana
The discovery of sulfated flavonoids in plants suggests that sulfation may play a regulatory role in the physiological functions of flavonoids. Sulfation of flavonoids is mediated by cytosolic sulfotransferases (SULTs), which utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. A novel SULT from Arabidopsis thaliana, designated AtSULT202B7 (AGI code: At1g13420), was cloned and expressed in Escherichia coli. Using various compounds as potential substrates, we demonstrated, for the first time, that AtSULT202B7 displayed sulfating activity specific for flavonoids. Intriguingly, the recombinant enzyme pre...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Hashiguchi, T., Sakakibara, Y., Shimohira, T., Kurogi, K., Yamasaki, M., Nishiyama, K., Akashi, R., Liu, M.-C., Suiko, M. Tags: Regular Papers Source Type: research
Biophysical characterization of the C-terminal region of FliG, an essential rotor component of the Na+-driven flagellar motor
In this study, using CD spectra, gel filtration chromatography and DSC (differential scanning calorimetry), we characterized the physical properties of the C-terminal domain (G214-Stop) of wild-type (WT) FliG and its non-motile phenotype mutant derivatives (L259Q, L270R and L271P), which were derived from the sodium-driven motor of Vibrio. The CD spectra and gel filtration chromatography revealed a slight difference between the WT and the mutant FliG proteins, but the DSC results suggested a large difference in their stabilities. That structural difference was confirmed by differences in protease sensitivity. Based on thes...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Gohara, M., Kobayashi, S., Abe-Yoshizumi, R., Nonoyama, N., Kojima, S., Asami, Y., Homma, M. Tags: Regular Papers Source Type: research
Strategy for cold adaptation of the tryptophan synthase {alpha} subunit from the psychrophile Shewanella frigidimarina K14-2: crystal structure and physicochemical properties
To investigate the molecular basis of cold adaptation of enzymes, we determined the crystal structure of the tryptophan synthase α subunit (SfTSA) from the psychrophile Shewanella frigidimarina K14-2 by X-ray analysis at 2.6-Å resolution and also examined its physicochemical properties. SfTSA was found to have the following characteristics: (i) The stabilities against heat and denaturant of SfTSA were lower than those of an α subunit (EcTSA) from Escherichia coli. This lower equilibrium stability originated from both a faster unfolding rate and a slower refolding rate; (ii) the heat denaturation of SfTSA ...
Source: Journal of Biochemistry - January 29, 2014 Category: Biochemistry Authors: Mitsuya, D., Tanaka, S.-i., Matsumura, H., Urano, N., Takano, K., Ogasahara, K., Takehira, M., Yutani, K., Ishida, M. Tags: Regular Papers Source Type: research
Intermolecular interactions and conformation of antibody dimers present in IgG1 biopharmaceuticals
Intermolecular interactions and conformation in dimer species of Palivizumab, a monoclonal antibody (IgG1), were investigated to elucidate the physical and chemical properties of the dimerized antibody. Palivizumab solution contains ~1% dimer and 99% monomer. The dimer species was isolated by size-exclusion chromatography and analysed by a number of methods including analytical ultracentrifugation-sedimantetion velocity (AUC-SV). AUC-SV in the presence of sodium dodecyl sulphate indicated that approximately half of the dimer fraction was non-covalently associated, whereas the other half was dimerized by covalent bond. Disu...
Source: Journal of Biochemistry - January 6, 2014 Category: Biochemistry Authors: Iwura, T., Fukuda, J., Yamazaki, K., Kanamaru, S., Arisaka, F. Tags: Regular Papers Source Type: research
Interaction of the phospholipid flippase Drs2p with the F-box protein Rcy1p plays an important role in early endosome to trans-Golgi network vesicle transport in yeast
Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574&nda...
Source: Journal of Biochemistry - January 6, 2014 Category: Biochemistry Authors: Hanamatsu, H., Fujimura-Kamada, K., Yamamoto, T., Furuta, N., Tanaka, K. Tags: Regular Papers Source Type: research
