(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...
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FB/Einrichtung: | FB 13: Biologie |
Dokumenttypen: | Artikel |
Medientypen: | Text |
Erscheinungsdatum: | 2014 |
Publikation in MIAMI: | 11.12.2014 |
Datum der letzten Änderung: | 16.04.2019 |
Angaben zur Ausgabe: | [Electronic ed.] |
Quelle: | AMB Express 4 (2014) 69, 1-9 |
Schlagwörter: | Fatty acid metabolism; 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase, Ralstonia eutropha H16 |
Fachgebiet (DDC): | 570: Biowissenschaften; Biologie |
Lizenz: | CC BY 4.0 |
Sprache: | English |
Anmerkungen: | Finanziert durch den Open-Access-Publikationsfonds 2014/2015 der Deutschen Forschungsgemeinschaft (DFG) und der Westfälischen Wilhelms-Universität Münster (WWU Münster). |
Format: | PDF-Dokument |
ISSN: | 2191-0855 |
URN: | urn:nbn:de:hbz:6-91319483386 |
Weitere Identifikatoren: | DOI: doi:10.1186/s13568-014-0069-0 |
Permalink: | https://nbn-resolving.de/urn:nbn:de:hbz:6-91319483386 |
Onlinezugriff: | s13568-014-0069-0.pdf |
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.