Purification and characterization of a Baeyer–Villiger mono-oxygenase from Rhodococcus erythropolis DCL14 involved in three different monocyclic monoterpene degradation pathways

2000 ◽  
Vol 347 (3) ◽  
pp. 693-701 ◽  
Author(s):  
Mariët J. VAN DER WERF
Keyword(s):  
Rhodococcus Erythropolis ◽  
Degradation Pathways ◽  
Rearrangement Product ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Prosthetic Group ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

A Baeyer-Villiger mono-oxygenase (BVMO), catalysing the NADPH- and oxygen-dependent oxidation of the monocyclic monoterpene ketones 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone, was purified to homogeneity from Rhodococcus erythropolis DCL14. Monocyclic monoterpene ketone mono-oxygenase (MMKMO) is a monomeric enzyme of molecular mass 60 kDa. It contains 1 mol of FAD/monomer as the prosthetic group. The N-terminal amino acid sequence showed homology with many other NADPH-dependent and FAD-containing (Type 1) BVMOs. Maximal enzyme activity was measured at pH 9 and 35 °C. MMKMO has a broad substrate specificity, catalysing the lactonization of a large number of monocyclic monoterpene ketones and substituted cyclohexanones. The natural substrates 1-hydroxy-2-oxolimonene, dihydrocarvone and menthone were converted stoichiometrically into 3-isopropenyl-6-oxoheptanoate (the spontaneous rearrangement product of the lactone formed by MMKMO), 4-isopropenyl-7-methyl-2-oxo-oxepanone and 7-isopropyl-4-methyl-2-oxo-oxepanone respectively. The MMKMO-catalysed conversion of iso-dihydrocarvone showed an opposite regioselectivity to that of dihydrocarvone; in this case, 6-isopropenyl-3-methyl-2-oxo-oxepanone was formed as the product. MMKMO converted all enantiomers of the natural substrates with almost equal efficiency. MMKMO is involved in the conversion of the monocyclic monoterpene ketone intermediates formed in the degradation pathways of all stereoisomers of three different monocyclic monoterpenes, i.e. limonene, (dihydro)carveol and menthol.

scholarly journals Purification and partial characterization of a lectin from Caesalpinia tinctoria Domb, ex Dc fruits

2003 ◽  
Vol 15 (2) ◽  
pp. 119-122 ◽  
Author(s):  
Marli Lourdes de Oliveira ◽  
Leila Maria Beltramini ◽  
Salvatore Giovanni de Simone ◽  
Maria Helena Nasser Brumano ◽  
Rosemeire Aparecida Silva-Lucca ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Terminal Amino Acid ◽  
Saline Extract ◽  
Hydrophobic Residues ◽  
Terminal Amino ◽  
Helix Conformation ◽  
Far Ultraviolet ◽  
Terminal Amino Acid Sequence

A lectin was isolated from the pod saline extract of Caesalpinia tinctoria by dialoconcentration on Centripep-10 and affinity chromatography on chitin column. The purified lectin was partially characterized with respect to its biochemical and structural properties. It contains 8.3 % of carbohydrate and exhibited an agglutinating activity against human erythrocytes (ABO groups). Its amino acid composition was characterized by a great number of acidic and hydrophobic residues and the estimated molecular mass was 12.5 kDa. The presence of only one N-terminal amino acid sequence (D¹-V-P-A-Y-V-Y-V-H-F10-G-F-G-E-E-H-R -D-V-F20-D), showed the homogeneity of the purified lectin. The far-ultraviolet circular dichroism (CD) spectrum of lectin indicated that it contains 10 % a-helix, 38 % b-sheet, 28 % unordered form and 6 % of P II (poly-L-proline II helix conformation).

scholarly journals Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1310-1322 ◽  
Author(s):  
MA De la Fuente ◽  
C Egile ◽  
A Pereira ◽  
M Juan ◽  
F Vivanco ◽  
...  
Keyword(s):  
Germ Line ◽  
Gene Segment ◽  
Cold Agglutinin ◽  
Functional Genes ◽  
Terminal Amino Acid ◽  
Clonal Origin ◽  
Vh Gene ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Abstract A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti- Gd specificity, found in a patient with Waldenstrom macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4–21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.

scholarly journals Purification and characterization of a previously unreported form of cytochrome P-448 from the liver of 3-methylcholanthrene-pretreated rats

1986 ◽  
Vol 235 (3) ◽  
pp. 859-868 ◽  
Author(s):  
S L Seidel ◽  
T K Shires
Keyword(s):  
Polyclonal Antibodies ◽  
The Other ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Spectral Maximum ◽  
Substrate Preferences ◽  
Terminal Amino ◽  
Cytochrome P 450

At least four hepatic isoenzymes of cytochrome P-450 were purified and characterized from rats treated with 3-methylcholanthrene. A monoclonal antibody developed against one of the forms (designated cytochrome P-450 MC-B) and polyclonal antibodies against others were used to demonstrate that form MC-B is immunologically distinct from other methylcholanthrene-inducible forms. Limited N-terminal amino acid sequencing showed that cytochrome P-450 MC-B has a primary structure that differs from the N-terminal sequences of other established rat isoenzymes. Cytochrome P-450 MC-B has a minimum Mr of 53,000, a CO-reduced spectral maximum at 448 nm, a Soret maximum of 417 nm in the absolute oxidized spectrum and a pattern of substrate preferences that differs from those of the other methylcholanthrene-induced forms. The other forms (MC-A, MC-C and MC-D) share characteristics with isoenzymes previously reported by other investigators.

scholarly journals TheN-terminal amino acid sequence of type 1 fimbria (pili) ofSalmonella typhimuriumLT2

1983 ◽  
Vol 16 (2-3) ◽  
pp. 149-151 ◽  
Author(s):  
Kristian Waalen ◽  
Knut Sletten ◽  
L.Oddvar Frøholm ◽  
Vuokko Väisänen ◽  
Timo K. Korhonen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture

2000 ◽  
Vol 46 (9) ◽  
pp. 856-859 ◽  
Author(s):  
Tong Li ◽  
Pierre Juteau ◽  
Réjean Beaudet ◽  
François Lépine ◽  
Richard Villemur ◽  
...  
Keyword(s):  
Gel Chromatography ◽  
Enterobacter Agglomerans ◽  
Anaerobic Conditions ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Cell Extracts ◽  
Sds Page ◽  
Oxygen Sensitive ◽  
Terminal Amino

The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity from a phenol-carboxylating coculture, consisting of Clostridium-like strain 6 and an unidentified strain 7, was studied. Assays done with cell extracts showed that the optimal pH was 5.0-6.5 and the Kmwas 5.4 mM. The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and even enhanced by the addition of Mg++, Mn++, Zn++, or Ca++. After purification, the molecular mass of the enzyme was estimated as 420 kDa by gel chromatography, and as 119 kDa by SDS-PAGE, suggesting a homotetrameric structure. Its pI was 5.6. The N-terminal amino acid sequence showed 95% and 76% homology with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from Enterobacter agglomerans and Klebsiella pneumoniae, respectively. The purified enzyme also slowly catalyzed the reverse reaction, that is the phenol carboxylation. These characteristics suggest that this enzyme is different from other known decarboxylases. This includes the 4OHB-DC from Clostridium hydroxybenzoicum, which is the only one that had been purified before.Key words: purification, 4-hydroxybenzoate decarboxylase, coculture, phenol carboxylation, anaerobic conditions.

scholarly journals Characterization of a monoclonal IgMK (IgMGAS) anti-Gd cold agglutinin (CA). Its coexistence with a monoclonal IgG3K (IgGGAS) without CA activity that might be clonally related to IgMGAS

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1310-1322
Author(s):  
MA De la Fuente ◽  
C Egile ◽  
A Pereira ◽  
M Juan ◽  
F Vivanco ◽  
...  
Keyword(s):  
Germ Line ◽  
Gene Segment ◽  
Cold Agglutinin ◽  
Functional Genes ◽  
Terminal Amino Acid ◽  
Clonal Origin ◽  
Vh Gene ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

A monoclonal IgMK (IgMGAS) cold agglutinin (CA) of the infrequent anti- Gd specificity, found in a patient with Waldenstrom macroglobulinemia, has been characterized. IgMGAS uses a VH gene homologous (93.7%) to the reported VH251 germ-line, one of the two functional genes of the VH5 family, with differences in both framework regions and complementary determining regions (CDR). The VL gene is homologous to the reported 15AVK1 germ-line gene, recently described in an anti-i CA, with differences mostly clustered in CDR. In the patient's serum, IgMGAS coexisted with a monoclonal IgG3K (IgGGAS) that lacked CA activity but expressed the private idiotopes found on IgMGAS. Both Ig lacked reactivity with antibodies detecting VH1 or VHIII or VKIII subgroup regions or the VH4–21 gene product that is expressed by anti-I/i CAs. The K chains from both Igs showed the same isoelectrical mobility. Moreover, the k chains from both serum Igs showed the same N-terminal amino acid sequence. This sequence was identical to that predicted by the nucleotide sequence of VK1GAS gene segment, including one discrepancy at position 15 (Ile for Val) with respect to the consensus VK1 subgroup regions. Although these data do not exclude a possible independent clonal origin, they are consistent with the notion that IgGGAS might be clonally related to IgMGAS.

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