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 Km value for acetoacetyl-CoA was 48 μM and Vmax 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.
Highlights
Ralstonia eutropha H16 is a Gram-negative β-proteobacterium
The expression of the β-oxidation genes in R. eutropha H16 is up-regulated, when the cells are grown in presence of fatty acids (FAs) (Brigham et al 2010)
Two operons located on chromosome #1 are responsible for the degradation of FAs in R. eutropha H16: H16_A0459-A0464 and H16_A1526-A1531
Summary
Ralstonia eutropha H16 is a Gram-negative β-proteobacterium. R. eutropha is able to utilize various fatty acids (FAs) as sole carbon source and to synthesize poly(3-hydroxybutyrate) (PHB) up to 80% or even more of its dry cell weight (Anderson and Dawes 1990). Two (S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratases, H16_A0461/FadB’ and H16_A1526/FadB1, are involved in the FA degradation in R. eutropha H16 (Brigham et al 2010). E. coli contains two fatty acid degradation (fad) operons. Their expression is induced when cells are grown in presence of FAs containing 12 or more carbon atoms (Klein et al 1971)
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