Purification and Properties of Human D-3-Hydroxyacyl-CoA Dehydratase: Medium-Chain Enoyl-CoA Hydratase Is D-3-Hydroxyacyl-CoA Dehydratase

Jiang, Ling; Kobayashi, Akio; Matsuura, Hirohide; Fukushima, Hirofumi; Hashimoto, Takashi

doi:10.1093/oxfordjournals.jbchem.a021458

Cited by 67 publications

(40 citation statements)

References 18 publications

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“…We performed a kinetic analysis on the purified enzyme measuring both kinetic and specific constant parameters in the direction of the hydratation towards crotonyl-CoA. K m value was 46.9 μM, very similar to other reported values for bacterial and mammals crotonases, e.g., C. acetobutylicum 30 μM (Bang et al 2001), E. coli 50 μM (Binstock and Schulz 1981), Homo sapiens 30 μM (Jiang et al 1996), and Sus scrofa 13 μM (Fong and Schulz 1977), confirming an analogue behavior for this ubiquitous enzyme. This evidence further suggests the identification of a real crotonyl-CoA activity.…”

Section: Methodssupporting

confidence: 79%

Bacillus subtilis fadB (ysiB) gene encodes an enoyl-CoA hydratase

et al. 2010

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Fatty acids are essential components of membranes and are an important source of metabolic energy. In bacteria, the β-oxidation pathway is well known in Escherichia coli. Bacillus subtilis possesses a considerable number of genes, organized in five operons, that are most likely involved in the β-oxidation of fatty acids. Among these genes, only one product, FadR Bs (YsiA), has been recently characterized as a transcriptional regulatory protein which negatively regulates the expression of β-oxidation genes including those belonging to the lcfA operon, including fadR Bs (ysiA). The probable involvement of the FadR Bs (YsiA) regulon members in β-oxidation is inferred from data based on BLASTP similarity of their gene products. In this work, we report the cloning and the expression of B. subtilis fadB Bs (ysiB), belonging to the lcfA operon, and the functional characterization of its product as an enoyl-CoA hydratase, demonstrating the actual involvement of these genes in fatty acid β-oxidation.

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Section: Methodssupporting

confidence: 79%

Bacillus subtilis fadB (ysiB) gene encodes an enoyl-CoA hydratase

et al. 2010

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“…The patients with MFE2 deficiency and MFE2 knock-out mice showed accumulations of VLCFA, branched chain fatty acids, and bile acid intermediates (39 -43). Jiang et al (44) reported that MFE2 purified from rat liver exhibited hydratase/ dehydrogenase activities on hexadecenoyl-CoA and 2-methylhexadecenoyl-CoA. In addition, although LCFA is largely degraded through the mitochondrial ␤-oxidation pathway, LCFA oxidation of MFE2-deficient human fibroblasts was lower than control fibroblasts under a condition in which the mitochondrial LCFA oxidation was inhibited (40).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Kashiwayama

Tomohiro

Narita

et al. 2010

Journal of Biological Chemistry

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Peroxisomes play an essential role in a number of important metabolic pathways including ␤-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various ␤-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal ␤-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp 249 to Arg 251 ) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp 249 to Arg 251 and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.Peroxisomes are organelles bound by a single membrane, which are present in almost all eukaryotic cells. Peroxisomes are involved in a variety of important metabolic processes including the ␤-oxidation of fatty acids, synthesis of plasmalogen, and bile acids (1). A specialized set of enzymes responsible for the peroxisomal functions are compartmentalized in the organelle, and the deficiency of peroxisomal enzymes causes severe metabolic disease such as acyl-CoA oxidase deficiency and D-bifunctional protein deficiency (2-4). Fibroblasts from patients with these deficiencies exhibit the disturbed peroxisomal ␤-oxidation of very long-chain fatty acids (VLCFAs), 4 and VLCFAs are accumulated in the plasma and tissues of these patients (5). Analyses of these peroxisomal diseases reflect the importance of the peroxisomal ␤-oxidation in organisms along with disclosing the enzymatic organization of the peroxisomal ␤-oxidation system. Peroxisomes are involved in the ␤-oxidation of VLCFAs, branched chain fatty acids such as pristanic acid, and bile acid intermediates such as dihydroxycholestanoic acid and trihydroxycholestanoic acid, which are incompatible with mitochondrial ␤-oxidation (6). Peroxisomal ␤-oxidation of fatty acids proceeds via a four-step pathway as in mitochondria, and multiple enzymes are involved in each step of the...

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“…We have reported the presence of the novel peroxisomaloxidation enzyme, d-BP (Jiang et al 1996a(Jiang et al , 1996b(Jiang et al , 1997a, and its congenital deficiency . l-BP deficiency was formerly considered to be the most frequent disorder among peroxisomal -oxidation enzyme defects (Watkins et al 1995).…”

Section: Discussionmentioning

confidence: 99%

“…d-BP converts enoyl-CoAs to 3-ketoacyl-CoAs via d-3-hydroxyacyl-CoAs in peroxisomes (Jiang et al 1996a(Jiang et al , 1996b. Peroxisomal -oxidation in human skin fibroblasts proceeds mainly by d-BP, but not by l-BP, as the content and activity of l-BP are very low in fibroblasts compared with that of d-BP (Jiang et al 1997a).…”

Section: Introductionmentioning

confidence: 99%