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Biomedical subjects

R Vicuña

Publications and source records attributed to R Vicuña.

34 records · Page 2Linked to original sources
PubMed1989

Benzaldehyde lyase, a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens biovar I.

Pseudomonas fluorescens biovar I can grow on benzoin as the sole carbon and energy source. This ability is due to benzaldehyde lyase, a new type of enzyme that irreversibly cleaves the acyloin linkage of benzoin, producing two molecules of benzaldehyde. Benzaldehyde lyase was purified 70-fold and found to require catalytic amounts of thiamine PPi (TPP) and a divalent cation as cofactors. Optimal activity was obtained with a 1.0 mM concentration of Mn2+, Mg2+, or Ca2+. Gel permeation chromatography indicated a native molecular weight of 80,000, whereas the enzyme migrated in sodium dodecyl sulfate-containing polyacrylamide gels as a single polypeptide with a molecular weight of 53,000. Benzaldehyde lyase is highly specific; of a variety of structurally related compounds tested, only benzoin and anisoin (4,4'-dimethoxybenzoin) acted as substrates, their apparent Kms being 9.0 x 10(-3) and 3.25 x 10(-2) mM, respectively. A catalytic mechanism for the enzyme is proposed.

Aldehyde-Lyases↗
PubMed1988

Degradation of diarylethane structures by Pseudomonas fluorescens biovar I.

Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4'-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported.

Alcohol Oxidoreductases↗
PubMed1988

Biochemical and genetic studies of bacteria metabolizing lignin-related compounds.

The ability of bacterial strains to metabolize lignin model compounds was studied. Strains examined were non-filamentous bacterial isolates obtained from decaying wood and the actinomycete Streptomyces viridosporus T7A. Model compounds included dimers containing either the beta-1 (1,2-diarylethane) or the beta-O-4 (arylglycerol-beta-aryl ether) type of linkage. Pseudomonas fluorescens biovar I A1 proliferated on anisoin (4,4'-dimethoxybenzoin) accumulating anisic acid temporarily. Cleavage at the beta-1 bond was also observed with crude extracts prepared from the same strain. In turn, cleavage of the beta-O-4 linkage of veratrylglycerol-beta-guaiacyl ether was detected in cultures of Pseudomonas acidovorans D3. In this case, main degradation intermediates were beta-hydroxypropioveratrone, acetoveratrone and guaiacol. S. viridosporus T7A reduced the carbonyl group of some beta-1 dimers and did not modify the beta-O-4 model compounds tested. Attempts to ascribe a catabolic character to large molecular weight extrachromosomal DNA present in some strains were unsuccessful. Gene banks of P. fluorescens biovar I A1 and P. acidovorans D3 were prepared utilizing the broad host range cosmid pLAFR1 as vector.

Bacteria↗
PubMed1987

Metabolism of Lignin Model Compounds of the Arylglycerol-beta-Aryl Ether Type by Pseudomonas acidovorans D(3).

A natural bacterial isolate that we have classified as Pseudomonas acidovorans grows on the lignin model compounds 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (compound 1'), as well as on the corresponding 1-oxo compounds (2 and 2') as sole sources of carbon and energy. Metabolic intermediates present in cultures growing on compound 1 included compound 2, 2-methoxyphenol (guaiacol [compound 3]), beta-hydroxypro-pioveratrone (compound 4), acetoveratrone (compound 5), and veratric acid (compound 6). Also identified were compounds 1', 2', beta-hydroxypropiovanillone (compound 4'), and acetovanillone (compound 5'), indicating that 4-O demethylation also occurs. The phenolic intermediates were the same as those found in cultures growing on compound 1'. Compounds 2 and 2' were in part also reduced to compounds 1 and 1', respectively. Compound 3 was shown to be derived from the 2-methoxyphenoxy moiety. A suggested degradation scheme is as follows: compound 1-->2-->(3 + 4)-->5-->6 (and similarly for 1'). In this scheme, the key reaction is cleavage of the ether linkage between C-2 (C(beta)) of the phenylpropane moiety and the 2-methoxyphenoxy moiety in compounds 2 and 2' (i.e., beta-aryl ether cleavage). On the basis of compounds identified, viz., 3 and 4 (4'), cleavage appears formally to be reductive. Because this is unlikely, the initial cleavage products probably were not detected. The implications of these results for the enzyme(s) responsible are discussed.

Journal Article↗
PubMed1985

DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8.

Three DNA polymerase isoenzymes, which have been called A, B and C, were purified from Thermus thermophilus HB-8. These enzymes can be separated by chromatography (pH 7.5) on phosphocellulose and DNA-agarose. Their relative molecular masses, as determined by glycerol gradient centrifugation, fall in the range of 110000-120000. The three of them are devoid of exonuclease activity. Species A, B and C differ in their sensitivity towards N-ethylmaleimide (A greater than B greater than C) and urea (A greater than B = C) and also in their stability at high temperature (90 degrees C) (B greater than C greater than A). In addition, these enzymes can be distinguished utilizing various templates under different conditions. Thus, with activated DNA and Mg2+ as a cofactor, the highest incorporation is obtained at 50 degrees C with enzyme A and at 63 degrees C with enzymes B and C. If Mg2+ is replaced by Mn2+, the optimal temperatures remain unchanged, but enzyme A is stimulated twofold, while the activities of enzymes B and C decrease to one-half. On the other hand, with either poly(dA) X (dT)10 or poly(dA-dT) and Mg2+, enzyme A is inactive and enzyme C is severalfold more active than enzyme B. With the former synthetic template, optimal temperatures are 50 degrees C (enzyme C) and 40 degrees C (enzyme B), while with poly(dA-dT) they both work best at 63 degrees C. In turn, only enzyme C is able to utilize poly(rA) X (dT)10, although only with Mn2+ as a cofactor.

Centrifugation, Density Gradient↗
PubMed1985

Electron microscopic mapping of Thermus thermophilus RNA polymerase binding sites on plasmid pBR322.

The binding of RNA polymerase from the extreme thermophile T. thermophilus HB8 to plasmid pBR322 was measured by electron microscopy. DNA-protein complexes were prepared at 35 and 60 degrees C. At both temperatures the enzyme binds strongly to sites which coincide with promoters P1, P2, P3 and P4 present in pBR322. At 60 degrees C, an additional binding site appears, which is located between P3 and P4. There is a high degree of correlation between RNA polymerase binding sites and the location of A-T rich regions on pBR322 DNA.

Binding Sites↗
PubMed1980

Deoxyribonucleic acid polymerase from the marine Pseudomonas sp. BAL-31.

A deoxyribonucleic acid (DNA)-dependent DNA polymerase (DNA nucleotidyltransferase) was purified 3,000-fold from the marine Pseuodomonas sp. BAL-31. The molecular weight of the native enzyme was estimated by glycerol gradient sedimentation to be 110,000. The enzyme migrated in sodium dodecyl sulfate-acrylamide gels as a single polypeptide with a molecular weight of 105,000. An absolute requirement for divalent cation was satisfied by Mg2+ or Mn2+ at concentrations of 1 mM. Monovalent cations at concentrations higher than 50 mM showed an inhibitory effect. The polymerase activity was resistant to N-ethylmaleimide and showed a wide pH optimum.

DNA-Directed DNA Polymerase↗