Limits...
(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

Neighbor-joining phylogenetic tree (Clustal X; Thompson et al.[1997]) based on amino acid sequence of different 3-hydroxyacyl-CoA dehydrogenases and the organization of FadB enzymes with different domain order (modified from Insomphun et al.[2014]): I- enzymes withE. coli-like structure; II- enzymes withBurkholderia-like structure. Accession numbers are given at the braces. Bar, 0.05 amino acid substitution per site.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230905&req=5

Figure 2: Neighbor-joining phylogenetic tree (Clustal X; Thompson et al.[1997]) based on amino acid sequence of different 3-hydroxyacyl-CoA dehydrogenases and the organization of FadB enzymes with different domain order (modified from Insomphun et al.[2014]): I- enzymes withE. coli-like structure; II- enzymes withBurkholderia-like structure. Accession numbers are given at the braces. Bar, 0.05 amino acid substitution per site.

Mentions: FadB’ consists of 806 amino acids (AA) with a theoretical molecular weight of 85.9 kDa and a pI of 7.66. The C terminus is annotated as a crotonase-like family/enoyl-CoA hydratase (ECH) domain and reveals similarity to the N-terminus of FadB1 (34% identity), a variety of enoyl-CoA hydratases like H16_B0389 (261 AA, 34% identity) and H16_B0987 (255 AA, 30% identity) from R. eutropha H16 and other organisms. The enoyl-CoA hydratase reaction is shown in Figure 1. The N-terminus of FadB’Re reveals similarities to genes of other 3-hydroxyacyl-CoA dehydrogenases of R. eutropha H16 like H16_A1102 (507 AA, 31% identity), H16_A0282 (284 AA, 33% identity), FadB1 (693 AA, 31% identity), and H16_B1652 (322 AA, 30% identity). This terminus comprises NAD binding (3HCDH_N) and 3HCDH domains and catalyzes the second step of the reaction shown in Figure 1. FadB1 is analogous to FadB’, although it possesses a reverse domain order (Insomphun et al. [2014]). Interestingly, a separate enoyl-CoA hydratase (H16_A0464, 11% identity) is located downstream of the H16_A0459-A0464 operon and reveals no significant similarity to the ECH-domain of FadB’Re. Amongst other bacteria most homologous to FadB’Re enzymes were found in many of species of the genus Burkholderia and other bacteria. Similarly organized 3-hydroxyacyl-CoA dehydrogenases were also found in Bacillus subtilis and Chromobacterium violaceum (Figure 2). According to the amino acid sequence and the order of domains two different groups of 3-hydroxyacyl-CoA dehydrogenases could be distinguished (Clustal X, Thompson et al. [1997]). The first group comprises two well-studied enzymes from E. coli, Pseudomonas mendocina and mammalians (as an example, Rattus norvegicus) with the so-called E. coli-like structure of 3-hydroxyacyl-CoA dehydrogenases. The representatives of the second group with an inverted order of the domains are less studied and are referred here to as Burkholderia-like enzymes.


(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)

Neighbor-joining phylogenetic tree (Clustal X; Thompson et al.[1997]) based on amino acid sequence of different 3-hydroxyacyl-CoA dehydrogenases and the organization of FadB enzymes with different domain order (modified from Insomphun et al.[2014]): I- enzymes withE. coli-like structure; II- enzymes withBurkholderia-like structure. Accession numbers are given at the braces. Bar, 0.05 amino acid substitution per site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Neighbor-joining phylogenetic tree (Clustal X; Thompson et al.[1997]) based on amino acid sequence of different 3-hydroxyacyl-CoA dehydrogenases and the organization of FadB enzymes with different domain order (modified from Insomphun et al.[2014]): I- enzymes withE. coli-like structure; II- enzymes withBurkholderia-like structure. Accession numbers are given at the braces. Bar, 0.05 amino acid substitution per site.
Mentions: FadB’ consists of 806 amino acids (AA) with a theoretical molecular weight of 85.9 kDa and a pI of 7.66. The C terminus is annotated as a crotonase-like family/enoyl-CoA hydratase (ECH) domain and reveals similarity to the N-terminus of FadB1 (34% identity), a variety of enoyl-CoA hydratases like H16_B0389 (261 AA, 34% identity) and H16_B0987 (255 AA, 30% identity) from R. eutropha H16 and other organisms. The enoyl-CoA hydratase reaction is shown in Figure 1. The N-terminus of FadB’Re reveals similarities to genes of other 3-hydroxyacyl-CoA dehydrogenases of R. eutropha H16 like H16_A1102 (507 AA, 31% identity), H16_A0282 (284 AA, 33% identity), FadB1 (693 AA, 31% identity), and H16_B1652 (322 AA, 30% identity). This terminus comprises NAD binding (3HCDH_N) and 3HCDH domains and catalyzes the second step of the reaction shown in Figure 1. FadB1 is analogous to FadB’, although it possesses a reverse domain order (Insomphun et al. [2014]). Interestingly, a separate enoyl-CoA hydratase (H16_A0464, 11% identity) is located downstream of the H16_A0459-A0464 operon and reveals no significant similarity to the ECH-domain of FadB’Re. Amongst other bacteria most homologous to FadB’Re enzymes were found in many of species of the genus Burkholderia and other bacteria. Similarly organized 3-hydroxyacyl-CoA dehydrogenases were also found in Bacillus subtilis and Chromobacterium violaceum (Figure 2). According to the amino acid sequence and the order of domains two different groups of 3-hydroxyacyl-CoA dehydrogenases could be distinguished (Clustal X, Thompson et al. [1997]). The first group comprises two well-studied enzymes from E. coli, Pseudomonas mendocina and mammalians (as an example, Rattus norvegicus) with the so-called E. coli-like structure of 3-hydroxyacyl-CoA dehydrogenases. The representatives of the second group with an inverted order of the domains are less studied and are referred here to as Burkholderia-like enzymes.

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