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A Bacher

Publications and source records attributed to A Bacher.

At least 217 records · Page 12Linked to original sources
PubMed1985

Structure of enoate reductase from a Clostridium tyrobutyricum (C. spec. La1).

Enoate reductase from Clostridium tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of about 90% pure enzyme in less than 10 h. A purity greater than 98% could be obtained by additional chromatography on Sephacryl S-300. The enzyme sediments in the analytical ultracentrifuge as a single, symmetrical boundary with a velocity of S(0)20,w = 24.9 S. Equilibrium ultracentrifugation yielded a molecular mass of 940 000 +/- 20 000 Da. The enzyme contains one type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit molecular mass of about 73 000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, iron and labile sulfur, the enzyme purified by the new method showed approximately 0.7 mol of FMN per mol of subunit. A dissociation product sedimenting at a velocity of S(0)20,w = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The molecular mass was 230 000 +/- 10 000 Da as shown by sedimentation equilibrium analysis. Thus it appears that the dissociation product is a trimer of the 73 000-Da subunit. The formation of the 10-S fragment by dissociation of the native enzyme is accompanied by the loss of most of the FMN, whereas the FAD content is not changed. The fragment catalysed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductase activity with NADH or methylviologen as cosubstrate was low. Electron micrographs of negatively stained enoate reductase show trigonal symmetry. The data suggest that enoate reductase is a dodecamer (tetramer of trimers) with tetrahedral symmetry.

Clostridium↗
PubMed1984

Biosynthesis of riboflavin. Enzymatic formation of the xylene moiety from [14C]ribulose 5-phosphate.

We have studied the enzymatic formation of the xylene ring of riboflavin using cell extracts from the flavinogenic yeast Candida guilliermondii. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate could serve as substrates. In addition, a pentose phosphate or pentulose phosphate was required. Experiments with [14C]ribulose 5-phosphate gave evidence for the incorporation of the ribulose carbon atoms except C-4 into the xylene ring of the vitamin.

Candida↗
PubMed1984

Separation of induction and expression of tight junction formation mediated by proteinases.

The formation of tight junctions can be induced in the human adenocarcinoma cell line HT 29 by treatment with trypsin at 37 degrees C. In contrast, after treatment of the cells with trypsin at low temperature (3 degrees C), no tight junctions were observed. However, abundant formation of tight junctions occurred when cells were treated with trypsin at 3 degrees C, washed with soybean trypsin inhibitor, and subsequently incubated at 37 degrees C. Thus, this protocol allows for the first time the temporal separation of the induction and assembly of tight junctions.

Adenocarcinoma↗
PubMed1983

Biosynthesis of riboflavin. Incorporation of 13C-labeled precursors into the xylene ring.

Growing cultures of Ashbya gossypii were supplemented with various 13C-labeled precursors including [1-13C]acetate, [2-13C]acetate, [1-13C]ribose, [1-13C]glucose, [6-13C]glucose, and [2-13C]glycerol. Riboflavin was isolated from the culture medium, chemically converted to riboflavin tetraacetate, and analyzed by 13C NMR spectroscopy. The xylene ring of riboflavin is formed by dismutation of 6,7-dimethyl-8-ribityllumazine, thus the 8 carbon atoms of the riboflavin xylene ring are composed of 4 biochemically different carbon atoms which are duplicated in the dismutation. The formation of the lumazine from a pyrimidine precursor requires the addition of these 4 carbon atoms which constitute C-6 alpha, C-6, C-7, and C-7 alpha of the lumazine. Results from the present work indicate that these 4 carbon atoms do not arise from acetate, diacetyl, acetoin, or a tetrose, nor from loss of 1 or 2 carbon atoms from either end of a pentose or hexose. Additionally, the data do not support the recent contention that these 4 atoms arise via a dismutation of the pyrimidine precursor of the lumazine. The findings show that there exists a close correspondence between the lumazine carbons 6 alpha, 6, and 7 with C-1, C-2, and C-3, respectively, of a pentose, while C-7 alpha corresponds to C-5 rather than C-4 of a pentose. This in conjunction with results reported earlier indicates an intramolecular rearrangement involving carbons 3, 4, and 5 of a pentose or its biochemical equivalent.

Acetates↗
PubMed1983

Crystallization and preliminary X-ray diffraction study of heavy riboflavin synthase from Bacillus subtilis.

Crystals of heavy riboflavin synthase from Bacillus subtilis have been obtained from 1.3 M sodium/potassium phosphate, pH 8.7, containing 0.35 mM 5-nitroso-6-(1'-D-ribitylamino)-2,4(1H,3H)-pyrimidinedione. X-ray photographs indicate a hexagonal unit cell with dimensions of a = b = 156.4 A; c = 298.5 A; alpha = beta = 90 degrees; gamma = 120 degrees. The space group was established as P6(3)22 with 12 general positions.

Bacillus subtilis↗
PubMed1983

Phosphates of riboflavin and riboflavin analogs: a reinvestigation by high-performance liquid chromatography.

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.

Apoproteins↗
PubMed1982

Ligand-binding studies on heavy riboflavin synthase of Bacillus subtilis.

Heavy riboflavin synthase is a complex enzyme consisting of three alpha subunits and approximately 60 beta subunits. Ligand-binding studies were performed with a variety of substrate and product analogues by analytical ultracentrifugation and by equilibrium dialysis. Nonlinear binding curves indicate the involvement of non-equivalent binding sites which could be assigned to the alpha and beta subunits by comparison with light riboflavin synthase (subunit composition alpha 3) and with aggregates of isolated beta subunits. The beta subunit binding site shows a high degree of stereospecificity. Tightly binding ligands must have a ribityl side chain and a pyrimidine or pteridine moiety with polar substituents.

Bacillus subtilis↗
PubMed1981

Biosynthesis of riboflavin. Characterization of the product of the deaminase.

The 2'5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate deaminase was partially purified from cell extracts of Candida guilliermondii ATCC 9058. The enzyme requires Mg2+ for activity. Maximal activity was observed at pH 7,3. The enzyme converts its substrate, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, to 2,5-diamino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate. This labile compound was treated with diacetyl and the resulting 6,7-dimethyl-8-ribityllumazine 5'-phosphate was identified by comparison with a synthetic sample.

Aminohydrolases↗
PubMed1981

Ligand-binding studies on light riboflavin synthase from Bacillus subtilis.

Light riboflavin synthase of Bacillus subtilis is a trimer of identical subunits. The enzyme catalyzes the transfer of a four-carbon moiety from one molecule of 6,7-dimethyl-8-ribityllumazine to a second molecule of this compound. Binding of substrate and product analogues to the enzyme was studied by analytical ultra-centrifugation and fluorescence titration. The ligands used in these experiments inhibit the enzyme activity competitively. Each enzyme subunit was shown to bind two molecules each of various analogues of the enzyme substrate, 6,7-dimethyl-8-ribityllumazine, at nonidentical sites. On the other hand, each subunit binds only one molecule of the product, riboflavin, or 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, an analogue of the second product. The complex of the enzyme with the substrate analogue, 7-methyl-8-ribityllumazine, was studied by absorbance and difference absorbance measurements. The data suggest that binding of the lumazine to the donor site of the enzyme involves a nucleophilic attack at carbon 7 of the lumazine ring with formation of a covalent hydrate or a related structure.

Bacillus subtilis↗
PubMed1980

Riboflavin synthases of Bacillus subtilis. Purification and properties.

A variety of Bacillus and Clostridium strains were found to contain two forms of riboflavin synthase which can be easily separated by density gradient centrifugation. The fast sedimenting species accounts for 12 to 44% of the total riboflavin synthase activity in the strains analyzed. Both riboflavin synthases were purified to apparent homogeneity from cell extracts of a genetically derepressed mutant of Bacillus subtilis. The specific activities of the pure proteins were 50,000 nmol mg-1 h-1 (light enzyme) and 2,000 nmol mg-1 h-1 (heavy enzyme). The sedimentation velocities (S20,w) were 4.1 and 26.5 S, respectively. Light riboflavin synthase showed a molecular weight of 70,000 in sedimentation equilibrium experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of about 23,500. Thus the enzyme appears to consist of three identical subunits (alpha type). Heavy riboflavin synthase has a molecular weight of 1,000,000 as shown by sedimentation equilibrium analysis. The protein appears to consist of 2 or 3 alpha subunits and approximately 60 beta subunits. A fragment apparently identical with light riboflavin synthase can be obtained from the heavy enzyme by mild dissociating treatment.

Bacillus subtilis↗
PubMed1978

Biosynthesis of riboflavin. 6,7-Dimethyl-8-ribityllumazine 5'-phosphate is not a substrate for riboflavin synthase.

Phosphotransferase from carrot is shown to catalyze the phosphorylation of 6,7-dimethyl-8-ribityllumazine specifically at position 5' of the ribityl side chain. The lumazine 5'-phosphate is neither a substrate nor an inhibitor of riboflavin synthase from Bacillus subtilis and Escherichia coli. It follows that the obligatory product of riboflavin synthase is riboflavin and not FMN.

Bacillus subtilis↗
PubMed1978

Biosynthesis of riboflavin in Bacillus subtilis: function and genetic control of the riboflavin synthase complex.

Two riboflavin synthase activities (heavy and light) have been observed in earlier studies with Bacillus subtilis. The heavy enzyme is a complex of one molecule of light enzyme (consisting of three alpha subunits) and approximately 60 beta subunits (A. Bacher, R. Bauer, U. Eggers, H. Harders, and H. Schnepple, p. 729--732, in T. P. Singer (ed.), Flavins and Flavoproteins, Elsevier, Amsterdam, 1976). The formation of alpha and beta subunits is coordinately controlled. Mutants apparently deficient in beta subunits were isolated as riboflavin requires after mutagenesis of B. subtilis with ICR 191. The mutants could grow with diacetyl instead of riboflavin. Growth with diacetyl was associated with the accumulation of substantial amounts of the riboflavin precursor, 6,7-dimethyl-8-(D-ribityl)lumazine. It follows that the mutants are deficient in an enzyme activity required for the formation of the lumazine from the pyrimidine precursor. We conclude that heavy riboflavin synthase is a bifunctional enzyme. The riboflavin synthase activity is mediated by the alpha subunits, whereas the beta subunits are necessary for an earlier biosynthetic step.

Bacillus subtilis↗
PubMed1977

Biosynthesis of thiamin in Bacillus subtilis. Isolation of mutants accumulating 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate.

Thiamin-deficient mutants of Bacillus subtilis were characterized by their growth responses to the pyrimidine and thiazole moieties of the vitamin molecule and by cross-feeding tests. All mutants growing on the thiazole moiety and all mutants with an absolute requirement for thiamin fed all those growing on the pyrimidine moiety. No other cross-feeding effects were observed. From the culture fluid of a mutant growing on the thiazole moiety, two compounds were isolated which supported growth of mutants requiring the pyrimidine moiety. These compounds were identified by chromatographic, bioautographic and spectrophotometric procedures as 4-amino-5-hydroxymethyl-2-methylpyrimidine and its monophosphate derivative.

Bacillus subtilis↗
PubMed1976

Biosynthesis of riboflavin. Structure of the purine precursor and origin of the ribityl side chain.

We studied the incorporation of 14C-labeled guanosine into riboflavin under conditions precluding the metabolic conversion of guanosine compounds to free guanine. For this purpose we isolated a mutant BM 2 of Salmonella typhimurium deficient in the enzymes IMP dehydrogenase, purine nucleoside phosphorylase, and purine nucleotide pyrophosphorylase. The mutant incorporated [ribose-14C]guanosine into riboflavin and GMP without dilution. The isolated compounds were exclusively labeled in the ribityl and ribosyl side chain, respectively. AMP and CMP were not labeled. [2-14C]Guanosine was incorporated into riboflavin and GMP without dilution. The isolated compounds were exclusively labeled in the isoalloxazine and guanine moiety, respectively. AMP and CMP were again unlabeled. We conclude that the ribose moiety of proffered guanosine is directly converted to the ribityl moiety of riboflavin. Thus the biosynthesis of the vitamin begins at the level of a guanosine compound. Guanine, ribose, ribitol, and the respective phosphates are not direct precursors of the vitamin.

Cell Division↗