Crystal structure of the atypical protein kinase domain of a TRP channel with phosphotransferase activity

Transient receptor potential (TRP) channels modulate calcium levels in eukaryotic cells in response to external signals. A novel transient receptor potential channel has the ability to phosphorylate itself and other proteins on serine and threonine residues. The catalytic domain of this channel kinase has no detectable sequence similarity to classical eukaryotic protein kinases and is essential for channel function. The structure of the kinase domain, reported here, reveals unexpected similarity to eukaryotic protein kinases in the catalytic core as well as to metabolic enzymes with ATP-grasp domains. The inclusion of the channel kinase catalytic domain within the eukaryotic protein kinase superfamily indicates a significantly wider distribution for this group of signaling proteins than suggested previously by sequence comparisons alone.     

Membrane-bound quinoprotein D-arabitol dehydrogenase of Gluconobacter suboxydans IFO 3257: a versatile enzyme for the oxidative fermentation of various ketoses

Solubilization of membrane-bound quinoprotein D-arabitol dehydrogenase (ARDH) was done successfully with the membrane fraction of Gluconobacter suboxydans IFO 3257. In enzyme solubilization and subsequent enzyme purification steps, special care was taken to purify ARDH as active as it was in the native membrane, after many disappointing trials. Selection of the best detergent, keeping ARDH as the holoenzyme by the addition of PQQ and Ca2+, and of a buffer system involving acetate buffer supplemented with Ca2+, were essential to treat the highly hydrophobic and thus labile enzyme. Purification of the enzyme was done by two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. ARDH was homogenous and showed a single sedimentation peak in analytical ultracentrifugation. ARDH was dissociated into two different subunits upon SDS-PAGE with molecular masses of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. ARDH was proven to be a quinoprotein by detecting a liberated PQQ from SDS-treated ARDH in HPLC chromatography. More preliminarily, an EDTA-treated membrane fraction lost the enzyme activity and ARDH activity was restored to the original level by the addition of PQQ and Ca2+. The most predominant unique character of ARDH, the substrate specificity, was highly versatile and many kinds of substrates were oxidized irreversibly by ARDH, not only pentitols but also other polyhydroxy alcohols including D-sorbitol, D-mannitol, glycerol, meso-erythritol, and 2,3-butanediol. ARDH may have its primary function in the oxidative fermentation of ketose production by acetic acid bacteria. ARDH contained no heme component, unlike the type II or type III quinoprotein alcohol dehydrogenase (ADH) and did not react with primary alcohols.     

Purification and characterization of membrane-bound quinoprotein cyclic alcohol dehydrogenase from Gluconobacter frateurii CHM 9

A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction for the first time, after extensive screening among aerobic bacteria. Gluconobacter frateurii CHM 9 was finally selected in this study. The enzyme tentatively named membrane-bound cyclic alcohol dehydrogenase (MCAD) was found to occur specifically in the membrane fraction, and pyrroloquinoline quinone (PQQ) was functional as the primary coenzyme in the enzyme activity. MCAD catalyzed only oxidation reaction of cyclic alcohols irreversibly to corresponding ketones. Unlike already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, MCAD was unable to catalyze the reverse reaction of cyclic ketones or aldehydes to cyclic alcohols. MCAD was solubilized and purified from the membrane fraction of the organism to homogeneity. Differential solubilization to eliminate the predominant quinoprotein alcohol dehydrogenase (ADH), and the subsequent two steps of column chromatographies, brought MCAD to homogeneity. Purified MCAD had a molecular mass of 83 kDa by SDS-PAGE. Substrate specificity showed that MCAD was an enzyme oxidizing a wide variety of cyclic alcohols. Some minor enzyme activity was found with aliphatic secondary alcohols and sugar alcohols, but not primary alcohols, differentiating MCAD from quinoprotein ADH. NAD-dependent cytosolic cyclic alcohol dehydrogenase (CCAD) in the same organism was crystallized and its catalytic and physicochemical properties were characterized. Judging from the catalytic properties of CCAD, it was apparent that CCAD was distinct from MCAD in many respects and seemed to make no contributions to cyclic alcohol oxidation.     

Binding site on human von Willebrand factor of bitiscetin,a snake venom-derived platelet aggregation inducer

Bitiscetin, a C-type lectin-like heterodimeric snake venom protein purified from Bitis arietans, binds to human von Willebrand factor (VWF) and induces the platelet membrane glycoprotein (GP) Ib-dependent platelet agglutination in vitro similar to botrocetin. In contrast with botrocetin which binds to the A1 domain of VWF, the A3 domain, a major collagen-binding site of VWF, was proposed to be a bitiscetin-binding site. In the competitive binding assay, neither bitiscetin nor botrocetin had an inhibitory effect on the VWF binding to the immobilized type III collagen on a plastic plate. The anti-VWF monoclonal antibody NMC-4, which inhibits VWF-induced platelet aggregation by binding to alpha4 helix of the A1 domain, also inhibited bitiscetin binding to the VWF. Binding of VWF to the immobilized bitiscetin was competitively inhibited by a high concentration of botrocetin. A panel of recombinant VWF, in which alanine-scanning mutagenesis was introduced to the charged amino acid residues in the A1 domain, showed that the bitiscetin-binding activity was reduced in mutations at Arg632, Lys660, Glu666, and Lys673 of the A1 domain. Those substituted at Arg629, Arg636, and Lys667, which decreased the botrocetin binding, showed no effect on the bitiscetin binding. These results indicate that bitiscetin binds to a distinct site in the A1 domain of VWF spanning over alpha4a, alpha5 helices and the loop between alpha5 and beta6 but close to the botrocetin- and NMC-4-binding sites. Monoclonal antibodies recognizing the alpha-subunit of bitiscetin specifically inhibited bitiscetin-induced platelet agglutination without affecting the binding between VWF and bitiscetin, suggesting that the alpha-subunit of bitiscetin is located on VWF closer to the GPIb-binding site than the beta-subunit is. Bitiscetin and botrocetin might modulate VWF by binding to the homologous region of the A1 domain to induce a conformational change leading to an increased accessibility to platelet GPIb.     

Activation of the lectin complement pathway by H-ficolin (Hakata antigen)

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.     

Changes in mutagenicity of herbicide chlornitrofen during biodegradation

We used the Ames assay to investigate changes in the mutagenicity of chlornitrofen during its aerobic biodegradation. Although a mixed culture of bacteria obtained from river water degraded chlornitrofen and reduced its concentration from 39 to 6 microg/l in 21 days, the indirect mutagenicity of the solution to Salmonella strains TA98, YG1021, and YG1026 increased gradually. This finding suggests that mutagenic metabolites were produced during the aerobic biodegradation. The increase in the mutagenicity was, however, much smaller under aerobic than under anaerobic conditions. The differing sensitivities of our test strains to the functional groups on the mutagens showed that the mutagenic metabolites were indirect frameshift-type mutagens that might have neither nitro nor amino groups.     

KIF1Bbeta2, capable of interacting with CHP,is localized to synaptic vesicles

Kinesin family proteins are microtubule-dependent molecular motors involved in the intracellular motile process. Using a Ca2+ -binding protein, CHP (calcineurin B homologous protein), as a bait for yeast two hybrid screening, we identified a novel kinesin-related protein, KIF1Bbeta2. KIF1Bbeta2 is a member of the KIF1 subfamily of kinesin-related proteins, and consists of an amino terminal KIF1B-type motor domain followed by a tail region highly similar to that of KIF1A. CHP binds to regions adjacent to the motor domains of KIF1Bbeta2 and KIF1B, but not to those of the other KIF1 family members, KIF1A and KIF1C. Immunostaining of neuronal cells showed that a significant portion of KIF1Bbeta2 is co-localized with synaptophysin, a marker protein for synaptic vesicles, but not with a mitochondria-staining dye. Subcellular fractionation analysis indicated the co-localization of KIF1Bbeta2 with synaptophysin. These results suggest that KIF1Bbeta2, a novel CHP-interacting molecular motor, mediates the transport of synaptic vesicles in neuronal cells.