Metabolism of lignin-related compounds by Rhodococcus rhodochrous: bioconversion of anisoin

Summary The ability of the nocardioform actinomycete Rhodococcus rhodochrous to metabolize selected lignin model compounds was studied. The compounds studied included cinnamic and ferulic acids and dimers possessing intermonomeric linkages that are characteristic of the lignin molecule. R. rhodochrous reduced the carbonyl group of anisoin, a 1,2-diarylethane (-1) structure to (1R,2R)-1,2-bis(4-methoxyphenyl)ethane-1,2-diol with an enantiomeric excess of .98%. Cleavage of 1,2-diarylethane and -O-4 structures by this strain could not be detected under our metabolic conditions. Offprint requests to: V. Andreoni     

Catabolism of 1,2-diarylethane lignin model compounds by two brown-rot fungi

The catabolism of dimethoxybenzil, anisoin and hydroanisoin in nitrogen-limited stationary cultures of the brown-rot fungi Wolfiporia cocos and Gloeophyllum trabeum was analyzed. These three 1,2-diarylethane lignin model compounds, which differ in the degree of oxidation of the alkylic chain, gave rise to p-anisaldehyde in both cultures, suggesting that cleavage between the two aliphatic carbons had occurred. In turn, both strains reduced dimethoxybenzil and anisoin to hydroanisoin, whereas only Wolfiporia cocos was able to oxidize hydro-anisoin to anisoin. On the other hand, chemically derived hydroxyl radical, but not superoxide radical, produced p-anisaldehyde plus other unidentified compounds from anisoin and hydroanisoin. Neither radical modified dimethoxybenzil significantly.Abbreviations HGLN high glucose, low nitrogen - HGHN high glucose, high nitrogen - HPLC high performance liquid chromatography - TLC thin layer chromatography - A anisoin - HA hydroanisoin - DMB dimethoxybenzil - OH Hydroxyl radical - O _inf2 ^sup- superoxide radical     

Utilization and cooxidation of chlorinated phenols byPseudomonas sp. B 13

Pseudomonas sp. B13 was grown in continuous culture on 4-chlorophenol as the only carbon source. Maximum growth rate of 0.4h^-1 was observed at a substrate concentration of >0.01 mM and <0.15 mM. In addition to the enzymes of phenol catabolism, high specific 1,2-dioxygenase activities with chlorocatechols as substrates were found. The isomeric monochlorinated phenols were also totally degraded by 4-chlorophenol grown cells. (+)-2,5-Dihydro-4-methyl- and (+)-2,5-dihydro-2-methyl-5-oxo-furan-2-acetic acid were formed in high yield as dead-end catabolites from cooxidation of cresoles.Several dichlorophenols except 2,6-dichlorophenol were removed from the culture fluid by chlorophenol grown cells. Ring cleavage of chlorinated catechols were shown to be one of the critical steps in chlorophenol catabolism. A catabolic pathway for isomeric chlorophenols is discussed.Non-Standard Abbreviations HPLC High performance liquid chromatography - DHB Dihydrodihydroxybenzoate 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid     

Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.     

Re-examination of cellular cyclic β-1,2-glucans of Rhizobiaceae: Distribution of ring sizes and degrees of glycerol-1-phosphate substitution

Gel-filtration and thin layer chromatography of low molecular weight carbohydrates from culture filtrates of Agrobacterium radiobacter, Isolate II, have shown, that next to the neutral -1,2-glucan fraction a major acidic fraction was present which was found to be glycerophosphorylated cyclic -1,2-glucans. Re-examination of cyclic -1,2-glucan preparations which had been obtained by extraction of Rhizobium cells with hot phenol-water also showed these acidic modified -1,2-glucans to be present. Cyclic -1,2-glucans from R. leguminosarum (9 strains) and of R. phaseoli (1 strain) had ring size distribution with degrees of polymerisation (DPs) of 19 and 20 as major ring sizes of which a minor part was glycerophosphorylated; -1,2-glucans of R. trifolii (3 strains) had ring sizes with DPs measuring 19–22 as prominent components which were largely unsubstituted, and R. meliloti (7 strains) had -1,2-glucans with ring size distributions extending to still higher DPs of 19–25 of which the major part appeared to be glycerophosphorylated.     

2-Hydroxychromene-2-carboxylate isomerase from bacteria that degrade naphthalenesulfonates

2-Hydroxychromene-2-carboxylate isomerase activity was found in cell-free systems from bacteria that degrade naphthalenesulfonates. The enzyme fromPseudomonas testosteroni A3 was activated by incubation with glutathione, dithiothreitol or mercaptoethanol. The highest enzyme activity was found after preincubation of the enzyme with glutathione at alkaline pH-values. A highly purified enzyme preparation converted besides 2-hydroxychromene-2-carboxylate also 2-hydroxybenzo[g]chromene-2-carboxylate (the 2-hydroxychromene-2-carboxylate formed from 1,2-dihydroxyanthracen). The addition of various metal ions or EDTA did not significantly change the catalytic activity of the enzyme. A possible reaction mechanism is proposed.Abbreviations 2,5-DHCCA 2,5-dihydroxychromene-2-carboxylate - 2,6-DHCCA 2,6-dihydroxychromene-2-carboxylate - 1,2-DHN 1,2-dihydroxynaphthalene - GSH glutathione - 2HBCCA 2-hydroxybenzo[g]chromene-2-carboxylate - HBP 2-hydroxybenzalpyruvate - HBPA 2-hydroxybenzalpyruvate aldolase - 2HCCA 2-hydroxychromene-2-carboxylate - 2HCCAI 2-hydroxychromene-2-carboxylate isomerase - 2NS naphthalene-2-sulfonate - R_t retention time     

Variations on the SnCl4 and CF3CO2Ag-promoted glycosidation of sugar acetates: a direct, versatile and apparently simple method with either α or β stereocontrol

Glycosidation of sugar peracetates (d-gluco, d-galacto) with SnCl₄ and CF₃CO₂Ag led to either 1,2-cis-, or 1,2-trans-glycosides, depending primarily on the alcohols used. In particular, 1,2-trans-glycosides, expected from acyl-protected glycosyl donors, were formed in high yields with alcohols sharing specific features such as bulkiness, presence of electron-withdrawing groups or polyethoxy motifs. In contrast, simple alcohols afforded 1:1 mixtures of 2,3,4,6-tetra-O-acetyl, and 3,4,6-tri-O-acetyl 1,2-cis-glycosides due to anomerization and/or acid-catalyzed fragmentation of 1,2-orthoester intermediates. After reacetylation or deacetylation, acetylated or fully deprotected 1,2-cis-glycosides (α-d-gluco, α-d-galacto) were obtained in 90% yields by a simple and direct method.     

Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome. Identification of two new structural genes of the glp-regulon

Summary A transducing phage carrying glpDlacZ, glpR, and malT was isolated from a strain harboring a glpDlacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpDlacZ and glpRlacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed     

Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H₂/CO₂ converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.Abbreviations BrES 2-bromoethanesulfonic acid - CA chloroethane - 1,2-DCA 1,2-dichloroethane - F₄₃₀ Ni(II)tetrahydro-(12, 13)-corphin with an uroporphinoid (III) ligand skeleton     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1

Pseudomonas paucimobilis Q1 originally isolated as biphenyl degrading organism (Furukawa et al. 1983), was shown to grow with naphthalene. After growth with biphenyl or naphthalene the strain synthesized the same enzyme for the ring cleavage of 2,3-dihydroxybiphenyl or 1,2-dihydroxynaphthalene. The enzyme, although characterized as 2,3-dihydroxybiphenyl dioxygenase (Taira et al. 1988), exhibited considerably higher relative activity with 1,2-dihydroxynaphthalene. These results demonstrate that this enzyme can function both in the naphthalene and biphenyl degradative pathway.Abbreviations DHBP dihydroxybiphenyl - DHBPDO 2,3-dihydroxybiphenyl dioxygenase - DHDHNDH 1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase - DHN 1,2-dihydroxynaphthalene - DHNDO 1,2-dihydroxynaphthalene dioxygenase - HBP cis-2-hydroxybenzalpyruvate - HOPDA 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate - PCB polychlorinated biphenyl - 2NS naphthalene-2-sulfonic acid     

Biosynthesis of cyclic β-(1,2)-glucans inRhizobium leguminosarum biovarsviciae,phaseoli andtrifolii

Inner membranes of Rhizobium leguminosarum biovars viciae, phaseoli, and trifolii synthesized a heterogenous family of neutral cyclic -(1,2)-glucans in a reaction system that used oligosaccharide intermediates covalently bound to a large protein. This glucoprotein showed a slightly lower mobility on SDS-polyacrylamide gels (apparent mol. mass of 320 kDa) than the -(1,2)-glucan intermediate protein of Rhizobium meliloti. In vivo pulse-label experiments with growing cells of R. leguminosarum biovar trifolii RS800 using radioactive glucose showed that few species of cyclic -(1,2)-glucans were synthesized and up to 30% were substituted with charged non-glycosidic residues, probably sn-1-phosphoglycerol.     

Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation

The chvB gene of Agrobacterium tumefaciens encodes a 235 kDa proteinaceous intermediate involved in the synthesis of -1,2-glucan. chvB mutants show a pleiotropic phenotype. Besides not to produce cyclic -1,2-glucan, chvB mutants have been reported to be avirulent, attachment-deficient, and nonmotile. In this study we report additional differences from the parent strain, probably all linked to changes in the cell envelope. This pleiotropic phenotype — except for attachment and virulence — could largely be prevented by growing chvB cells with low levels of calcium. Although a role for -1,2-glucan in osmoadaptation has been proposed, the mode of action of -1,2-glucan is not known. We speculate that in A. tumefaciens -1,2-glucan stabilizes membranes, which would be important especially in hypotonic media containing calcium.Abbreviations Cb carbenicillin - Km kanamycin - TCA trichloroacetic acid - K_av fraction of the stationary gel volume available for diffusion - LPS lipopolysaccharide - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Pyruvate fermentation in Rhodospirillum rubrum and after transfer from aerobic to anaerobic conditions in the dark

The fermentative metabolism of Rhodospirillum rubrum (strain Ha, F₁, S₁) was studied after transfering the cells from aerobic to anaerobic dark culture conditions. Pyruvate was metabolized mainly to acetate and formate, and to a lesser extent to CO₂ and propionate, by all strains. Therefore, pyruvate formate lyase would appear to be the characteristic key enzyme of the dark anaerobic fermentation metabolism in R. rubrum. Strain F₁ and S₁ metabolized the formate further to H₂ and CO₂. It is concluded that this cleavage was catalysed by a formate hydrogen lyase system. Strain Ha was unable to metabolize formate. The cleavage of formate and the synthesis of poly--hydroxy-butyric acid were increased by a low pH value (6.5). Fermentation equations and schemes of the pyruvate metabolism are discussed.     

Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5′-AT/TAAT-3′

Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI. This enzyme was isolated from cell extracts by phosphocellulose chromatography. Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5-AT/TAAT-3 generating 5 dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of VspI, a restriction enzyme isolated from Vibrio sp.     

Degradation of d,l-syringaresinol,a β-β′ linked lignin model compound,by Fusarium solani M-13-1

A - linked lignin model compound, d,l-syringaresinol monobenzyl ether (Ib) was incubated with Fusarium solani M-13-1 in a shaking culture. From the culture filtrates, three compounds II, IIIb and IV were isolated and identified. Substrate Ib was oxidized at the -position of the side chain to give a hemiketal, an -hydroxylated compound IIA, which was then transformed to the ketoalcohol, 3-hydroxymethyl-2-(4-benzyloxy-3,5-dimethoxyphenyl)-4-(4-hydroxy-3,5-dimethoxybenzoyl)-tetrahydrofuran (IIB). These products were converted to a -lactone derivative, 6-oxo-2-(4-benzyloxy-3,5-dimethoxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane (IIIb), via alkyl-aryl cleavage. The syringyl moiety released from II by the cleavage reaction was identified as 2,6-dimethoxy-p-benzoquinone (IV). Incubation of 2,6-dimethoxyphenol (V) in fungal culures did not give the p-quinone IV. d,l-Syringaresinol dimethyl ether was not degraded and the etherated moiety of Ib was not attacked by the fungus, indicating that the degradation of d,l-syringaresinol was catalyzed by phenol oxidizing enzymes. The oxidation products of Ib with peroxidase/H₂O₂ was investigated and discussed in relation to the degradation products of the fungus.Abbreviation TLC thin layer chromatography     

Differential accumulation of four phaseolin glycoforms in transgenic tobacco

An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical -phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (wt^i–) and mutant phaseolin glycoforms (dgly ₁, dgly ₂ and dgly _1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the dgly ₁ and dgly ₂ mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin dgly _1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.Abbreviations bp base pair(s) - DAF days after flowering - GUS -glucuronidase - kb kilobase - kDa kilodalton