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(S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB') from fatty acid degradation operon of Ralstonia eutropha H16.

Volodina E, Steinbüchel A - AMB Express (2014)

Bottom Line: FadB' was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD(+).FadB' exhibited optimal activity at pH 6-7 and the activity decreased at alkaline and acidic pH values.Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB'.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, Münster, D-48149, Germany.

ABSTRACT
In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB', gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB' is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB' comprises an NAD(+) binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB' was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD(+). The K m value for acetoacetyl-CoA was 48 μM and V max 149 μmol mg(-1) min(-1). NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB' exhibited optimal activity at pH 6-7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB'. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB' and fatty acid metabolism, FadB' might be also interesting for biotechnological applications.

No MeSH data available.


Related in: MedlinePlus

Dependence of the inhibition of FadB’ activity on the concentration of acetyl-CoA, propionyl-CoA and CoA. The dotted lines demonstrate the inhibitory effect on FadB’ activity measured with acetoacetyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl buffer (pH 7.0) 0.1 mM acetoacetyl-CoA, 0.2 mM NADH, 0.3 μg FadB’Re and the inhibitor CoA or acetyl-CoA at the concentrations as indicated. The straight lines demonstrate the inhibitory effect on FadB’ measured with crotonyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl (pH 8.1) 0.1 mM crotonyl-CoA, 2 mM pyruvate, lactate dehydrogenase (9 U), 1.5 mM NAD+, 25 mM MgCl2 and the inhibitor CoA, acetyl-CoA or propionyl-CoA at the concentrations as indicated. All values are presented as mean values of at least two measurements, standard deviations varied between 0.01% and 3.58%.
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Figure 3: Dependence of the inhibition of FadB’ activity on the concentration of acetyl-CoA, propionyl-CoA and CoA. The dotted lines demonstrate the inhibitory effect on FadB’ activity measured with acetoacetyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl buffer (pH 7.0) 0.1 mM acetoacetyl-CoA, 0.2 mM NADH, 0.3 μg FadB’Re and the inhibitor CoA or acetyl-CoA at the concentrations as indicated. The straight lines demonstrate the inhibitory effect on FadB’ measured with crotonyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl (pH 8.1) 0.1 mM crotonyl-CoA, 2 mM pyruvate, lactate dehydrogenase (9 U), 1.5 mM NAD+, 25 mM MgCl2 and the inhibitor CoA, acetyl-CoA or propionyl-CoA at the concentrations as indicated. All values are presented as mean values of at least two measurements, standard deviations varied between 0.01% and 3.58%.

Mentions: Enoyl-CoA hydratase activity of FadB’ was inhibited by 70% in presence of 0.1 mM acetyl-CoA and by 30% in presence of 0.1 mM CoA (Figure 3). Propionyl-CoA was tested additionally to verify the inhibitory effect. Similarly, as with acetyl-CoA about 60% of the FadB’ activity was lost in presence of 0.1 mM propionyl-CoA. Higher concentrations of the mentioned CoA-thioesters and CoA inhibited the FadB’ activity stronger. 0.5 mM propionyl-CoA reduced the activity of FadB’ to 28% and 0.5 mM acetyl-CoA resulted in only 7% of residual activity. However, FadB’ retained 7% of the initial activity in presence of 1 mM acetyl-CoA and 11% - in presence of propionyl-CoA.


(S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB') from fatty acid degradation operon of Ralstonia eutropha H16.

Volodina E, Steinbüchel A - AMB Express (2014)

Dependence of the inhibition of FadB’ activity on the concentration of acetyl-CoA, propionyl-CoA and CoA. The dotted lines demonstrate the inhibitory effect on FadB’ activity measured with acetoacetyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl buffer (pH 7.0) 0.1 mM acetoacetyl-CoA, 0.2 mM NADH, 0.3 μg FadB’Re and the inhibitor CoA or acetyl-CoA at the concentrations as indicated. The straight lines demonstrate the inhibitory effect on FadB’ measured with crotonyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl (pH 8.1) 0.1 mM crotonyl-CoA, 2 mM pyruvate, lactate dehydrogenase (9 U), 1.5 mM NAD+, 25 mM MgCl2 and the inhibitor CoA, acetyl-CoA or propionyl-CoA at the concentrations as indicated. All values are presented as mean values of at least two measurements, standard deviations varied between 0.01% and 3.58%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230905&req=5

Figure 3: Dependence of the inhibition of FadB’ activity on the concentration of acetyl-CoA, propionyl-CoA and CoA. The dotted lines demonstrate the inhibitory effect on FadB’ activity measured with acetoacetyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl buffer (pH 7.0) 0.1 mM acetoacetyl-CoA, 0.2 mM NADH, 0.3 μg FadB’Re and the inhibitor CoA or acetyl-CoA at the concentrations as indicated. The straight lines demonstrate the inhibitory effect on FadB’ measured with crotonyl-CoA as substrate. The reaction mixture contained in 100 mM Tris–HCl (pH 8.1) 0.1 mM crotonyl-CoA, 2 mM pyruvate, lactate dehydrogenase (9 U), 1.5 mM NAD+, 25 mM MgCl2 and the inhibitor CoA, acetyl-CoA or propionyl-CoA at the concentrations as indicated. All values are presented as mean values of at least two measurements, standard deviations varied between 0.01% and 3.58%.
Mentions: Enoyl-CoA hydratase activity of FadB’ was inhibited by 70% in presence of 0.1 mM acetyl-CoA and by 30% in presence of 0.1 mM CoA (Figure 3). Propionyl-CoA was tested additionally to verify the inhibitory effect. Similarly, as with acetyl-CoA about 60% of the FadB’ activity was lost in presence of 0.1 mM propionyl-CoA. Higher concentrations of the mentioned CoA-thioesters and CoA inhibited the FadB’ activity stronger. 0.5 mM propionyl-CoA reduced the activity of FadB’ to 28% and 0.5 mM acetyl-CoA resulted in only 7% of residual activity. However, FadB’ retained 7% of the initial activity in presence of 1 mM acetyl-CoA and 11% - in presence of propionyl-CoA.

Bottom Line: FadB' was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD(+).FadB' exhibited optimal activity at pH 6-7 and the activity decreased at alkaline and acidic pH values.Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB'.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, Münster, D-48149, Germany.

ABSTRACT
In this study (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (H16_A0461/FadB', gene ID: 4247876) from one of two active fatty acid degradation operons of Ralstonia eutropha H16 has been heterologously expressed in Escherichia coli, purified as protein possessing a His-Tag and initially characterized. FadB' is an enzyme with two catalytic domains exhibiting a single monomeric structure and possessing a molecular weight of 86 kDa. The C-terminal part of the enzyme harbors enoyl-CoA hydratase activity and is able to convert trans-crotonyl-CoA to 3-hydroxybutyryl-CoA. The N-terminal part of FadB' comprises an NAD(+) binding site and is responsible for 3-hydroxyacyl-CoA dehydrogenase activity converting (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA. Enoyl-CoA hydratase activity was detected spectrophotometrically with trans-crotonyl-CoA. (S)-3-Hydroxyacyl-CoA dehydrogenase activity was measured in both directions with acetoacetyl-CoA and 3-hydroxybutyryl-CoA. FadB' was found to be strictly stereospecific to (S)-3-hydroxybutyryl-CoA and to prefer NAD(+). The K m value for acetoacetyl-CoA was 48 μM and V max 149 μmol mg(-1) min(-1). NADP(H) was utilized at a rate of less than 10% in comparison to activity with NAD(H). FadB' exhibited optimal activity at pH 6-7 and the activity decreased at alkaline and acidic pH values. Acetyl-CoA, propionyl-CoA and CoA were found to have an inhibitory effect on FadB'. This study is a first report on biochemical properties of purified (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase with the inverted domain order from R. eutropha H16. In addition to fundamental information about FadB' and fatty acid metabolism, FadB' might be also interesting for biotechnological applications.

No MeSH data available.


Related in: MedlinePlus