(S)-3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase (FadB') from fatty acid degradation operon of Ralstonia eutropha H16
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 char...
|Division/Institute:||FB 13: Biologie|
|Date of publication on miami:||11.12.2014|
|Edition statement:||[Electronic ed.]|
|Source:||AMB Express 4 (2014) 69, 1-9|
|Subjects:||Fatty acid metabolism; 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase, Ralstonia eutropha H16|
|DDC Subject:||570: Biowissenschaften; Biologie|
|License:||CC BY 4.0|
|Notes:||Finanziert durch den Open-Access-Publikationsfonds 2014/2015 der Deutschen Forschungsgemeinschaft (DFG) und der Westfälischen Wilhelms-Universität Münster (WWU Münster).|
|Other Identifiers:||DOI: doi:10.1186/s13568-014-0069-0|
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.