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Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity.

Jin J, Straathof AJ, Pinkse MW, Hanefeld U - Appl. Microbiol. Biotechnol. (2010)

Bottom Line: A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773.The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation.The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters.

View Article: PubMed Central - PubMed

Affiliation: Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. j.jin@tudelft.nl

ABSTRACT
A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters.

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UV–visible spectrum of purified 3-hydroxycyclohexanone dehydrogenase (MhyADH). The spectrum of the oxidized enzyme (2.4 μM) was recorded in 20 mM Tris–HCl, pH 7.8. The inset shows the spectra of oxidized (solid line) and reduced enzyme (dotted line). The reduction of the enzyme was performed by addition of 1 mM 3-hydroxycyclohexanone
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Fig4: UV–visible spectrum of purified 3-hydroxycyclohexanone dehydrogenase (MhyADH). The spectrum of the oxidized enzyme (2.4 μM) was recorded in 20 mM Tris–HCl, pH 7.8. The inset shows the spectra of oxidized (solid line) and reduced enzyme (dotted line). The reduction of the enzyme was performed by addition of 1 mM 3-hydroxycyclohexanone

Mentions: The purified MhyADH showed absorption maxima in the UV region at 271 nm, in the visible region at 440 nm, and broad shoulders around 350 and 550 nm (Fig. 4), corresponding to the spectra of other molybdoflavin proteins including ketone dehydrogenase and nicotinic acid dehydrogenase (Schräder et al. 2002; Baitsch et al. 2001). Cofactor content was determined by coupled plasma-mass spectrometry (ICP-MS) and catalytic AdSV. Metal analysis by ICP-MS showed that the purified enzyme contained molybdenum, iron, and zinc in a ratio of 1:4.05:0.8. A similar ratio of 1: 4.6: 1.1 was reported in the study of nicotinic acid dehydrogenase (Schräder et al. 2002). Molybdenum was determined by AdSV, and the result showed that one monomer contains 1.19 ± 0.15 Mo/protein molecule. The optimum pH and thermal stability of MhyADH were determined. MhyADH has a bell-shaped pH profile with 80% activity between pH 6.5 and 8.0. The enzyme showed the highest activity at pH 7.5. In the potassium phosphate buffer at pH 7.5, MhyADH showed a half-life of ~120 min at 50°C but is quickly deactivated after 15 min at 70°C.Fig. 4


Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity.

Jin J, Straathof AJ, Pinkse MW, Hanefeld U - Appl. Microbiol. Biotechnol. (2010)

UV–visible spectrum of purified 3-hydroxycyclohexanone dehydrogenase (MhyADH). The spectrum of the oxidized enzyme (2.4 μM) was recorded in 20 mM Tris–HCl, pH 7.8. The inset shows the spectra of oxidized (solid line) and reduced enzyme (dotted line). The reduction of the enzyme was performed by addition of 1 mM 3-hydroxycyclohexanone
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044224&req=5

Fig4: UV–visible spectrum of purified 3-hydroxycyclohexanone dehydrogenase (MhyADH). The spectrum of the oxidized enzyme (2.4 μM) was recorded in 20 mM Tris–HCl, pH 7.8. The inset shows the spectra of oxidized (solid line) and reduced enzyme (dotted line). The reduction of the enzyme was performed by addition of 1 mM 3-hydroxycyclohexanone
Mentions: The purified MhyADH showed absorption maxima in the UV region at 271 nm, in the visible region at 440 nm, and broad shoulders around 350 and 550 nm (Fig. 4), corresponding to the spectra of other molybdoflavin proteins including ketone dehydrogenase and nicotinic acid dehydrogenase (Schräder et al. 2002; Baitsch et al. 2001). Cofactor content was determined by coupled plasma-mass spectrometry (ICP-MS) and catalytic AdSV. Metal analysis by ICP-MS showed that the purified enzyme contained molybdenum, iron, and zinc in a ratio of 1:4.05:0.8. A similar ratio of 1: 4.6: 1.1 was reported in the study of nicotinic acid dehydrogenase (Schräder et al. 2002). Molybdenum was determined by AdSV, and the result showed that one monomer contains 1.19 ± 0.15 Mo/protein molecule. The optimum pH and thermal stability of MhyADH were determined. MhyADH has a bell-shaped pH profile with 80% activity between pH 6.5 and 8.0. The enzyme showed the highest activity at pH 7.5. In the potassium phosphate buffer at pH 7.5, MhyADH showed a half-life of ~120 min at 50°C but is quickly deactivated after 15 min at 70°C.Fig. 4

Bottom Line: A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773.The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation.The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters.

View Article: PubMed Central - PubMed

Affiliation: Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. j.jin@tudelft.nl

ABSTRACT
A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,β-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters.

Show MeSH