ラットにおける脂質代謝および胆石に及ぼす大豆純化不齢化物の効果

The influences of the purified unsaponifiable fraction of soybean (PUFS) on gallstone formation and dissolution and related lipid metabolism were investigated in mice fed lithogenic diet in comparison with soybean sterols (SBST).　　In experiment I, the effect of each drug on gallstone formation was studied during 8 weeks. All of mice fed lithogenic diet had a large number of gallstones in gallbladder but PUFS treatment inhibited markedly the stone production. The influence of SBST alone was weaker than PUFS itself. In this experiment, the serum and liver cholesterol levels were parallel to the degree of gallstone production. The liver enlarged three times as large as control under the lithogenic condition was improved with PUFS or SBST. In experiment II, the effect of drugs on gallstone dissolution was investigated. The mice already having gallstone after feeding a lithogenic diet for 8 weeks were kept on the normal chow with or without PUFS for further 5 weeks. The tendency to dissolve gallstone was not observed in both groups but serum and liver cholesterol contents and liver weight returned more rapidly to normal level in the PUFS treated group. As experiment III, the mice already having gallstone were continuously maintained on the lithogenic diet with or without PUFS during 6 weeks. No effect on gallstone dissolution was found but the desirable influences on lipid metabolism were also clearly observed

MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of <i>Tetrahymena thermophila</i>

<div><p>To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in <i>Bacillus subtilis</i>). It has been proposed that the MtnBD fusion enzyme in <i>Tetrahymena thermophila</i> catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in <i>B. subtilis</i>) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from <i>B. subtilis</i>. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of <i>T. thermophila</i> MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.</p></div

UV-visible and ¹H-NMR spectra of metabolites synthesized by <i>Tetrahymena</i> MtnBD.

<p>(A) Conversion of MTRu-1-P into HK-MTPenyl-1-P in D₂O phosphate buffer. Dehydration of MTRu-1-P (upper part) may introduce a proton from solvent into C4 of HK-MTPenyl-1-P (lower part). (B) UV-visible spectral changes after adding MtnBD into reaction mixture containing MTRu-1-P. Spectra are shown in different colors (0, 40, 80, 120, 160, 200, 240, 300, and 420 s). (C) Spectral changes before and after adding 0.14 µg <i>B. subtilis</i> MtnX to reaction product in Fig. 2B. Spectra are shown in different colors (0, 10, 20, 30, 40, 50, 60, 80, 100, and 220 s). In (B) and (C), spectra of products were measured at 35°C in 100 µL 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 100 µM MTR-1-P, and 9 µg <i>Bacillus</i> MtnA. MTR-1-P was converted into MTRu-1-P by MtnA before assay. (D) Time course of HK-MTPenyl-1-P production with different amounts of MtnBD protein. The reaction was initiated by adding 0.7, 1.5 or 2.2 µg MtnBD proteins to 100 µL 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 100 µM MTR-1-P, and 9 µg MtnA at 35°C. MTR-1-P was converted into MTRu-1-P by MtnA before assay. Concentration of HK-MTPenyl-1-P was estimated using the molecular extinction coefficient (9,500 M⁻¹ cm⁻¹ at 280 nm). (E) ¹H-NMR spectrum of product (MTRu-1-P) from <i>B. subtilis</i> MtnA. Isomerization reaction in 250 µL 25 mM sodium phosphate (pD 7.5), 0.1 mM MgCl₂, 1 mM MTR-1-P, and 20 µg <i>B. subtilis</i> MtnA at 37°C for 30 min. (F to H) ¹H-NMR spectra after adding 10 µg MtnBD into reaction mixture containing MTRu-1-P. * indicates chemical shift associated with degraded compound (elimination of methyl group at C1). Reaction products generated in magnesium phosphate buffer at 25°C and pD 7.5 at 1 h (F), 2 h (G) and 6 h (H). ¹H peak at 4.74 ppm represents proton from residual H₂O.</p

Methionine salvage pathway (MSP).

<p>(A) MSP in <i>B. subtilis</i> and other organisms. Enzyme names are derived from Ashida et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067385#pone.0067385-Ashida2" target="_blank">[19]</a> and Salim et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067385#pone.0067385-Salim2" target="_blank">[14]</a>. The MSP recycles organic sulfur from MTA, a by-product of polyamine synthesis, to methionine. MTA phosphorylase (MtnP) in mammals and yeasts catalyzes a one-step reaction to yield MTR-1-P from MTA. In almost all microorganisms, plants, and specific protozoa except for <i>T. thermophila</i>, this phosphorylase is replaced by two enzymes; MTA nucleosidase (MtnN) and MTR kinase (MtnK). (B) Diversity of MSP enzymes among various organisms. In <i>B. subtilis</i>, the MSP consists of eight enzymes because it has one enzyme for each reaction. While <i>B. subtilis</i> utilizes two separate enzymes (MtnW and MtnX) to catalyze the enolization and dephosphorylation steps, in most living organisms including proteobacteria, yeasts, plants, and animals, DK-MTP-1-P is converted into HK-MTPenyl-1-P by a bi-functional DK-MTP-1-P enolase/phosphatase (MtnC) belonging to the haloacid dehalogenase superfamily. In plants and <i>T. thermophila,</i> MtnB is fused with MtnC and MtnD, respectively.</p

Sequence, structure, and phylogenetic analyses of various MtnBs.

<p>(A) Multiple sequence alignment of MtnBs. Magenta triangles indicate histidine residues essential for binding divalent metal ion. Numbers on left of sequences show innate amino acid number for each protein. The alignment was created using ClustalW; identical and similar amino acids were highlighted/shaded with Boxshade. (B) Predicted tertiary structure of the <i>T. thermophila</i> MtnB domain. This was predicted from the known structure of <i>A. aeolicus</i> MtnB (PDB ID: 2IRP) using Swiss-model (<a href="http://swissmodel.expasy.org/" target="_blank">http://swissmodel.expasy.org/</a>). Three histidine residues essential for metal binding are shown by magenta sticks. Nitrogen atoms of histidine residues at active site are shown in blue. Structure was drawn using PyMOL version 0.98 (<a href="http://pymol.org" target="_blank">http://pymol.org</a>). (C) Phylogenetic tree based on primary sequences of MtnB family. Alignments were created with ClustalW and displayed using Treeview. Scale bar indicates difference of 0.1 substitutions per site. Full names and gene accession numbers are as follows: <i>Anopheles gambiae</i> str. PEST (XP_310624), <i>A. aeolicus</i> VF5 (NP_214357), <i>Arabidopsis thaliana</i> (NP_974931 residues 1–247), <i>Aspergillus nidulans</i> FGSC A4 (XP_661197), <i>B. subtilis</i> str 168 (NP_389244), <i>Caenorhabditis elegans</i> (NP_509690), <i>Chlorella variabilis</i> (EFN54454 residues 1–254), <i>Cryptococcus neoformans</i> var. <i>neoformans</i> JEC21 (XP_572402), <i>Danio rerio</i> (NP_001004679), <i>Dictyostelium discoideum</i> (XP_639930), <i>Drosophila melanogaster</i> (NP_572916), <i>Enterobacter cloacae</i> subsp. <i>cloacae</i> ATCC 13047 (YP_003613571), <i>Geobacillus kaustophilus</i> HTA426 (YP_146808), <i>Homo sapiens</i> (NP_057041), <i>Hydra magnipapillata</i> (XP_002165198), <i>Klebsiella oxytoca</i> (formerly <i>Klebsiella pneumoniae</i> 342) (YP_002239745), <i>Microcystis aeruginosa</i> PCC7806 (CAO89699), <i>Monosiga brevicollis</i> MX1 (XP_001750472), <i>Mus musculus</i> (NP_062709), <i>Neurospora crassa</i> OR74A (XP_964699), <i>Oryza sativa</i> Japonica Group (NP_001067908 residues 1–252), <i>Pseudomonas aeruginosa</i> PAO1 (NP_250374), <i>Saccharomyces cerevisiae</i> S288c (NP_012558), <i>Schizosaccharomyces pombe</i> 972h- (NP_593625), <i>Strongylocentrotus purpuratus</i> (XP_794552 residues 3114–3362), <i>Synechococcus elongatus</i> PCC 6301 (YP_172813), <i>Vitis vinifera</i> (XP_002274553 residues 1–257), <i>Volvox carteri</i> f. <i>nagariensis</i> (XP_002956646 residues 1–218), <i>Xenopus tropicalis</i> (NP_001015712). Although some MtnBs consist of more than two domains, only MtnB domains were used for phylogenetic analysis. Proteins described in (A) are underscored.</p

HPLC analyses of reaction products of dioxygenase reaction.

<p>(A) Authentic KMTB (1 mM). (B) Chromatogram of products generated by MtnBD and <i>B. subtilis</i> MtnX. The triangle represents KMTB. * indicates an unidentified peak at 11.8 min.</p