Characterization of an Inducible Chlorophenol O-Methyltransferase from Trichoderma longibrachiatum Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines

ABSTRACT A novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-3H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine. Gel filtration indicated that the M r was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The Km values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 μM, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a Ki of 378.9 ± 45.4 μM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu2+, Hg2+, Zn2+, and Ag+, and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.

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Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

Biochemistry and Cell Biology ◽

10.1139/o86-169 ◽

1986 ◽

Vol 64 (12) ◽

pp. 1288-1293 ◽

Cited By ~ 6

Author(s):

Josefa M. Alonso ◽

Amando Garrido-Pertierra

Keyword(s):

Pseudomonas Putida ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Competitive Inhibitor ◽

Catabolic Pathway ◽

Ph Optimum ◽

Semialdehyde Dehydrogenase ◽

Standard Assay ◽

Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

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Purification, Characterization, and Cloning of a Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Key Enzyme Involved in Biosynthesis of Terpenoids

Journal of Bacteriology ◽

10.1128/jb.181.4.1256-1263.1999 ◽

1999 ◽

Vol 181 (4) ◽

pp. 1256-1263 ◽

Cited By ~ 42

Author(s):

Shunji Takahashi ◽

Tomohisa Kuzuyama ◽

Haruo Seto

Keyword(s):

Amino Acid ◽

Coenzyme A ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Ph Optimum ◽

Hmg Coa Reductase ◽

Apparent Homogeneity ◽

Key Enzyme ◽

Coa Reductase

ABSTRACT The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34 ) was purified 3,000-fold fromStreptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 μM for NADPH and 7.7 μM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.

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Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis

Biochemical Journal ◽

10.1042/bj1990795 ◽

1981 ◽

Vol 199 (3) ◽

pp. 795-805 ◽

Cited By ~ 11

Author(s):

A H J Ullah ◽

G W Ordal

Keyword(s):

Bacillus Subtilis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Single Species ◽

Competitive Inhibitor ◽

Sensory Stimuli ◽

Molecular Weights ◽

Protein Methyltransferase

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.

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Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.252-256.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 252-256 ◽

Cited By ~ 19

Author(s):

Katsuichi Saito ◽

Kazuya Kondo ◽

Ichiro Kojima ◽

Atsushi Yokota ◽

Fusao Tomita

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Extracellular Enzyme ◽

Molecular Weights ◽

Sds Page ◽

Monomeric Structure ◽

Ph Range ◽

Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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Characterization of proteins from the cytoskeleton of Giardia lamblia

Journal of Cell Science ◽

10.1242/jcs.59.1.81 ◽

1983 ◽

Vol 59 (1) ◽

pp. 81-103 ◽

Cited By ~ 1

Author(s):

R. Crossley ◽

D.V. Holberton

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulphate ◽

Giardia Lamblia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Molecular Size ◽

Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

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Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

Zeitschrift für Naturforschung C ◽

10.1515/znc-1996-5-611 ◽

1996 ◽

Vol 51 (5-6) ◽

pp. 342-354 ◽

Cited By ~ 9

Author(s):

Beate Nicolaus ◽

Yukiharu Sato ◽

Ko Wakabayashi ◽

Peter Böger

Keyword(s):

Protein Pattern ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Total Activity ◽

Naphthalic Anhydride ◽

Molecular Weights ◽

Isomerase Activity ◽

Ammonium Sulfate Precipitate ◽

Catalyzed Reactions

Abstract Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

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Purification, characterization and inhibition of human skin collagenase

Biochemical Journal ◽

10.1042/bj1690265 ◽

1978 ◽

Vol 169 (2) ◽

pp. 265-276 ◽

Cited By ~ 89

Author(s):

David E. Woolley ◽

Robert W. Glanville ◽

Dennis R. Roberts ◽

John M. Evanson

Keyword(s):

Human Skin ◽

Culture Medium ◽

Serum Proteins ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Collagen Fibrils ◽

Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

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Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4

Journal of Bacteriology ◽

10.1128/jb.01418-06 ◽

2007 ◽

Vol 189 (7) ◽

pp. 2660-2666 ◽

Cited By ~ 19

Author(s):

Vandana P. Swetha ◽

Aditya Basu ◽

Prashant S. Phale

Keyword(s):

Molecular Mass ◽

High Performance ◽

Polyacrylamide Gel Electrophoresis ◽

Substrate Inhibition ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Sole Source ◽

Maximum Activity ◽

Ph Optimum ◽

Phenol Derivatives

ABSTRACT Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.

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Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

Biochemistry and Cell Biology ◽

10.1139/o87-116 ◽

1987 ◽

Vol 65 (10) ◽

pp. 899-908 ◽

Cited By ~ 21

Author(s):

F. Moranelli ◽

M. Yaguchi ◽

G. B. Calleja ◽

A. Nasim

Keyword(s):

Amino Acid ◽

Amylase Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Deae Cellulose ◽

Exchange Chromatography ◽

Proteinase K ◽

Ph Optimum ◽

Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

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Cathepsin D from pig myometrium. Characterization of the proteinase

Biochemical Journal ◽

10.1042/bj2190899 ◽

1984 ◽

Vol 219 (3) ◽

pp. 899-904 ◽

Cited By ~ 12

Author(s):

R Barth ◽

E G Afting

Keyword(s):

Cathepsin D ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Polyacrylamide Gel ◽

Ph Optimum ◽

Equilibrium Studies ◽

Main Band ◽

Km Value ◽

Ph Range

The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.

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