Purification and properties of phloroglucinol reductase from Eubacterium oxidoreducens G-41

Phloroglucinol reductase was purified 90-fold to homogeneity from the anaerobic rumen organism Eubacterium oxidoreducens strain G-41. The enzyme is stable in the presence of air and is found in the soluble fraction after ultracentrifugation of cell extract. Ion-exchange, hydrophobic interaction, and affinity chromatography were used to purify the enzyme. The native Mr is 78,000, and the subunit Mr is 33,000 indicating an alpha 2 homodimer. The enzyme is specific for phloroglucinol and NADPH. The Km and Vmax are 600 microM and 640 mumol min-1 mg-1 (pH 7.2) for phloroglucinol, and 6.7 microM and 550 mumol min-1 mg-1 (pH 6.8) for NADPH; the Km and Vmax for the reverse direction are 290 microM and 140 mumol min-1 mg-1 (pH 7.2) for dihydrophloroglucinol, and 27 microM and 220 mumol min-1 mg-1 (pH 7.2) for NADP. Temperature and pH optima are 40 degrees C and 7.8 in the forward direction. The pure enzyme is colorless in solution and flavins are absent. Analysis for cobalt, manganese, molybdenum, vanadium, tungsten, selenium, copper, nickel, iron, and zinc indicated that these metals are not components of the phloroglucinol reductase. Cupric chloride, n-ethylmaleimide, and p-chloromercuribenzoate are potent inhibitors of enzyme activity. The properties of phloroglucinol reductase indicate that it functions in the pathway of anaerobic degradation of trihydroxybenzenes by catalyzing reduction of the aromatic nucleus prior to ring fission.     

Metabolism of gallate and phloroglucinol in Eubacterium oxidoreducens via 3-hydroxy-5-oxohexanoate

The pathway for the anaerobic catabolism of gallic acid by Eubacterium oxidoreducans was studied by using both in vivo and cell-free systems. Cells grown with gallate and crotonate, but with no formate or H2, excreted pyrogallol and phloroglucinol into the medium. Gallate was decarboxylated by crude cell extracts, with pyrogallol as the only detectable product. Whole cells converted pyrogallol to phloroglucinol. A phloroglucinol reductase catalyzed the conversion of phloroglucinol to dihydrophloroglucinol when NADPH was used as the source of electrons. Both formate dehydrogenase (EC 1.2.1.43) and hydrogenase (EC 1.18.99.1) were present in cell extracts of gallate-formate-grown cells. These two enzymes were both NADP linked. Since either H2 or formate is required for cell growth with gallate or phloroglucinol, these results suggest that the oxidation of the reduced substrate may be indirectly linked to the reduction of phloroglucinol. A dihydrophloroglucinol hydrolase was present, which hydrolyzed dihydrophloroglucinol to 3-hydroxy-5-oxohexanoate. This six-carbon ring cleavage product then presumably can be broken down by a series of reactions similar to beta-oxidation. These reactions cleaved the six-carbon acid to 3-hydroxybutyryl-coenzyme A yielding acetate and butyrate as end products. A number of key enzymes involved in beta-oxidation and substrate-level phosphorylation were demonstrated in cell extracts.     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Initial steps in the anaerobic degradation of 3,4,5-trihydroxybenzoate by Eubacterium oxidoreducens: characterization of mutants and role of 1,2,3,5-tetrahydroxybenzene.

Chemical mutagenesis and antibiotic enrichment techniques were used to isolate five mutant strains of the obligate anaerobe Eubacterium oxidoreducens that were unable to grow on 3,4,5-trihydroxybenzoate (gallate). Two strains could not transform gallate and showed no detectable gallate decarboxylase activity. Two other strains transformed gallate to pyrogallol and dihydrophloroglucinol but lacked the hydrolase activity responsible for ring cleavage. A fifth strain accumulated pyrogallol, although it contained adequate levels of the enzymes proposed for the complete transformation of gallate to the ring cleavage product. The conversion of pyrogallol to phloroglucinol by cell extract of the wild-type strain was dependent on the addition of 1,2,3,5-tetrahydroxybenzene or dimethyl sulfoxide. This activity was induced by growth on gallate, while the other enzymes involved in the initial reactions of gallate catabolism were constitutively expressed during growth on crotonate. The results confirm the initial steps in the pathway previously proposed for the metabolism of gallate by E. oxidoreducens, except for the conversion of pyrogallol to phloroglucinol.     

Transformation of (sup14)C-Lignin-Labeled Cell Walls of Wheat by Syntrophococcus sucromutans, Eubacterium oxidoreducens, and Neocallimastix frontalis

Wheat cell walls, saponified or not, labeled with [U-(sup14)C]phenylalanine or [O-methyl-(sup14)C]sinapate were fermented by Neocallimastix frontalis or Syntrophococcus sucromutans plus Eubacterium oxidoreducens or a mixed culture. Phenolics were less solubilized but more transformed by bacteria than by the fungus, and mineralization was slight. S. sucromutans O-demethylated [O-methyl-(sup14)C]syringyl lignins, yielding labeled acetate.     

Characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus and Eubacterium aerofaciens.

Peptostreptococcus productus strain b-52 (a human fecal isolate) and Eubacterium aerofaciens ATCC 25986 were found to contain NADP-dependent 7 beta-hydroxysteriod dehydrogenase activity. The enzyme was synthesized constitutively by both organisms, and the enzyme yields were suppressed by the addition of 0.5 mM 7 beta-hydroxy bile acid to the growth medium. Purification of the enzyme by chromatography resulted in preparations with 3.5 (P. productus b-52, on Sephadex G-200) and 1.8 (E. aerofaciens, on Bio-Gel A-1.5 M) times the activity of the crude cell extracts. A pH optimum of 9.8 and a molecular weight of approximately 53,000 were shown for the enzyme of strain b-52, and an optimum pH at 10.5 and a molecular weight of 45,000 was shown for that from strain ATCC 25986. Kinetic studies revealed that both enzyme preparations oxidized the 7 beta-hydroxy group in unconjugated and conjugated bile acids, a lower Km value being demonstrated with free bile acid than with glycine and taurine conjugates. No measureable activity against 3 alpha-, 7 alpha-, or 12 alpha-hydroxy groups was detected in either enzyme preparation. When tested with strain ATCC 25986, little 7 beta-hydroxy-steroid dehydrogenase activity was detected in cells grown in the presence of glucose in excess. The enzyme from strain b-52 was found to be heat labile (90% inactivation at 50 degrees C for 3 min) and highly sensitive to sulfhydryl inhibitors.     

Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase.

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.     

Characterization of Aspergillus fumigatus protein disulfide isomerase family gene.

Aspergillus fumigatus is an opportunistic fungus which causes pulmonary complications in humans and animals. The clinical spectrum observed with A. fumigatus is attributed to the multifunctional nature of its antigens. Lack of understanding on the molecular processes and complexity of the fungus have spurred interest in the identification and characterization of its antigens/allergens with biological activities and virulence functions. For identification of some of these antigens/allergens, a cDNA library of A. fumigatus was screened with antibodies of allergic bronchopulmonary aspergillosis (ABPA) patients. One of the reactive clones was sequenced and observed to have an open reading frame of 1095 nucleotides corresponding to a polypeptide of 364 amino acids. The nucleotide and deduced amino acid sequence showed significant homology with the protein disulfide isomerase (PDI) superfamily. The expressed recombinant fusion protein exhibited specific IgG and IgE binding with antibodies present in ABPA patients' sera. The recombinant protein in vitro catalyzed folding of scrambled RNase. The probable epitopic regions of the deduced amino acid sequence were mapped by algorithmic analysis. This is the first report of isolation of a gene encoding a member of the PDI family from A. fumigatus. The PDI superfamily of proteins may play an important role in the protein folding mechanisms of A. fumigatus antigens/allergens for their interaction with the host.     

Characterization of the S-denitrosation activity of protein disulfide isomerase

S-nitrosoglutathione (GSNO) denitrosation activity of recombinant human protein disulfide isomerase (PDI) has been kinetically characterized by monitoring the loss of the S-NO absorbance, using a NO electrode, and with the aid of the fluorogenic NOx probe 2,3-diaminonaphthalene. The initial rates of denitrosation as a function of [GSNO] displayed hyperbolic behavior irrespective of the method used to monitor denitrosation. The Km values estimated for GSNO were 65 +/- 5 microm and 40 +/- 10 microm for the loss in the S-NO bond and NO production (NO electrode or 2,3-diaminonaphthalene), respectively. Hemoglobin assay provided additional evidence that the final product of PDI-dependent GSNO denitrosation was NO*. A catalytic mechanism, involving a nitroxyl disulfide intermediate stabilized by imidazole (His160 a-domain or His589 a'-domain), which after undergoing a one-electron oxidation decomposes to yield NO plus dithiyl radical, has been proposed. Evidence for the formation of thiyl/dithiyl radicals during PDI-catalyzed denitrosation was obtained with 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl. Evidence has also been obtained showing that in a NO- and O2-rich environment, PDI can form N2O3 in its hydrophobic domains. This "NO-charged PDI" can perform intra- and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Interestingly, reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO. PDI-SNO, once formed, is stable at room temperature in the absence of reducing agent over the period of 2 h. It has been established that PDI is continuously secreted from cells that are net producers of NO-like endothelial cells. The present demonstration that PDI can be S-nitrosated and that PDI-SNO can be denitrosated by PDI suggests that this enzyme could be intimately involved in the transport of intracellular NO equivalents to the cell surface as well as the previous demonstration of PDI in the transfer of S-nitrosothiol-bound NO to the cytosol.     

Characterization of acid-induced unfolding intermediates of glucose/xylose isomerase.

Acid-induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1-anilino-8-naphthalene-sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule-like characteristics, such as the presence of native-like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N-->PFI-->MG-->U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 M GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric alpha/beta barrel protein.     

New markers for Eubacterium lentum.

Of 37 strains of Eubacterium lentum and phenotypically similar organisms, 26 (70%) synthesized a corticoid 21-dehydroxylase and/or a 3 alpha-hydroxysteroid dehydrogenase. It appeared that the corticoid 3 alpha-hydroxysteroid dehydrogenase was identical to the bile acid 3 alpha-hydroxysteroid dehydrogenase. Steroid-metabolizing enzymes were found both in E. lentum and in phenotypically similar organisms. E. lentum is characterized by nitrate reduction and enhanced growth in the presence of arginine. Many phenotypically similar organisms possess either one or the other of the two markers. In contrast, using the steroid-metabolizing enzymes as markers, a "steroid-active" and a "steroid-inactive" group were established with minimal overlapping of metabolic characteristics. Synthesis of the steroid enzymes was positively correlated with production of gas from H2O2 and formation of H2S. A simple method for the detection of corticoid 21-dehydroxylase and 3 alpha-hydroxysteroid dehydrogenase, one or both of which were present in 92% of the steroid-active group, is described.     

Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene

The gene that codes for xylose isomerase in Escherichia coli has been cloned by complementation of a xylose isomerase-negative E. coli mutant. The structural gene is 1320 nucleotides in length and codes for a protein of 440 amino acids. An additional 209 nucleotides 5' and 82 nucleotides 3' to the structural gene were also sequenced. To verify that the cloned gene encodes E. coli xylose isomerase, the enzyme was purified to homogeneity and the sequence of the first 25 amino acid residues was determined by a semimicromanual Edman procedure. These results establish that the NH2-terminal methionine of xylose isomerase is specified by an ATG which is 7 nucleotides downstream from a Shine-Dalgarno sequence.     

Towards the reaction mechanism of pyrogallol-phloroglucinol transhydroxylase of Pelobacter acidigallici

Conversion of pyrogallol to phloroglucinol was studied with the molybdenum enzyme transhydroxylase of the strictly anaerobic fermenting bacterium Pelobacter acidigallici. Transhydroxylation experiments in H218O revealed that none of the hydroxyl groups of phloroglucinol was derived from water, confirming the concept that this enzyme transfers a hydroxyl group from the cosubstrate 1,2,3, 5-tetrahydroxybenzene (tetrahydroxybenzene) to the acceptor pyrogallol, and simultaneously regenerates the cosubstrate. This concept requires a reaction which synthesizes the cofactor de novo to maintain a sufficiently high intracellular pool during growth. Some sulfoxides and aromatic N-oxides were found to act as hydroxyl donors to convert pyrogallol to tetrahydroxybenzene. Again, water was not the source of the added hydroxyl groups; the oxides reacted as cosubstrates in a transhydroxylation reaction rather than as true oxidants in a net hydroxylation reaction. No oxidizing agent was found that supported a formation of tetrahydroxybenzene via a net hydroxylation of pyrogallol. However, conversion of pyrogallol to phloroglucinol in the absence of tetrahydroxybenzene was achieved if little pyrogallol and a high amount of enzyme preparation was used which had been pre-exposed to air. Obviously, the enzyme was oxidized by air to form sufficient amounts of tetrahydroxybenzene from pyrogallol to start the reaction. A reaction mechanism is proposed which combines an oxidative hydroxylation with a reductive dehydroxylation via the molybdenum cofactor, and allows the transfer of a hydroxyl group between tetrahydroxybenzene and pyrogallol without involvement of water. With this, the transhydroxylase differs basically from all other hydroxylating molybdenum enzymes which all use water as hydroxyl source.     

Characterization of an L-arabinose isomerase from Bacillus subtilis

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k _cat of 14,504 min⁻¹ and a k _cat/K _m of 121 min⁻¹ mM⁻¹ for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose.     

Characterization of Sinorhizobium meliloti triose phosphate isomerase genes

A Tn5 mutant strain of Sinorhizobium meliloti with an insertion in tpiA (systematic identifier SMc01023), a putative triose phosphate isomerase (TPI)-encoding gene, was isolated. The tpiA mutant grew more slowly than the wild type on rhamnose and did not grow with glycerol as a sole carbon source. The genome of S. meliloti wild-type Rm1021 contains a second predicted TPI-encoding gene, tpiB (SMc01614). We have constructed mutations and confirmed that both genes encode functional TPI enzymes. tpiA appears to be constitutively expressed and provides the primary TPI activity for central metabolism. tpiB has been shown to be required for growth with erythritol. TpiB activity is induced by growth with erythritol; however, basal levels of TpiB activity present in tpiA mutants allow for growth with gluconeogenic carbon sources. Although tpiA mutants can be complemented by tpiB, tpiA cannot substitute for mutations in tpiB with respect to erythritol catabolism. Mutations in tpiA or tpiB alone do not cause symbiotic defects; however, mutations in both tpiA and tpiB caused reduced symbiotic nitrogen fixation.     

Growth of cholesterol-reducing Eubacterium on cholesterol-brain agar.

An agar medium containing 5% cholesterol has been developed for the isolation, enumeration, and characterization of cholesterol-reducing strains of Eubacterium.