Expression and purification of His-tagged rat mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase wild-type and Ser137 mutant proteins

Mitochondrial 3-hydroxyacyl-CoA dehydrogenase is a key enzyme in the beta-oxidation of fatty acids. The deficiency of this enzyme in patients has been previously reported. We cloned the gene of rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase in a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' of the gene. The cloned gene was overexpressed in Escherichia coli and the soluble protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity. The specific activity of the purified His-tagged rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase was 452 U/mg. Ser137 is a highly conserved amino acid, which, it has been suggested, is an important residue because of its proximity to the modeled L-3-hydroxyacyl-CoA substrate in the crystal structure of 3-hydroxyacyl-CoA dehydrogenase. We constructed three mutant expression plasmids of the enzyme using site-directed mutagenesis. Mutant proteins were overexpressed in E. coli and purified with a nickel metal affinity column. Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that Ser137 is a very important residue of rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase. Our overexpression in E. coli and one-step purification of the highly active rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of 3-hydroxyacyl-CoA dehydrogenase.     

Recognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/amyloid-beta binding alcohol dehydrogenase (ABAD)

Human type II hydroxyacyl-CoA dehydrogenase/amyloid-beta binding alcohol dehydrogenase (HADH II/ABAD) is an oxidoreductase whose salient features include broad substrate specificity, encompassing 3-hydroxyacyl-CoA derivatives, hydroxysteroids, alcohols and beta-hydroxybutyrate, and the capacity to bind amyloid-beta peptide, leading to propagation of amyloid-induced cell stress. In this study, we examine the structure and enzymatic activity of the homologous rat HADH II/ABAD enzyme. We report the crystal structure of rat HADH II/ABAD as a binary complex with its NADH cofactor to 2.0 A resolution, as a ternary complex with NAD(+) and 3-ketobutyrate (acetoacetate) to 1.4 A resolution, and as a ternary complex with NADH and 17 beta-estradiol to 1.7 A resolution. This first crystal structure of an HADH II confirms these enzymes are closely related to the short-chain hydroxysteroid dehydrogenases and differ substantially from the classic, type I 3-hydroxyacyl-CoA dehydrogenases. Binding of the ketobutyrate substrate is accompanied by closure of the active site specificity loop, whereas the steroid substrate does not appear to require closure for binding. Despite the different chemical nature of the two bound substrates, the presentation of chemical groups within the active site of each complex is remarkably similar, allowing a general mechanism for catalytic activity to be proposed. There is a characteristic extension to the active site that is likely to accommodate the CoA moiety of 3-hydroxyacyl-CoA substrates. Rat HADH II/ABAD also binds amyloid-beta (1-40) peptide with a K(D) of 21 nM, which is similar to the interaction exhibited between this peptide and human HADH II/ABAD. These studies provide the first structural insights into HADH II/ABAD interaction with its substrates, and indicate the relevance of the rodent enzyme and associated rodent models for analysis of HADH II/ABAD's physiologic and pathophysiologic properties.     

D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder.

Peroxisomal beta-oxidation proceeds from enoyl-CoA through D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA by the D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxy-acyl-CoA dehydrogenase bifunctional protein (d-bifunctional protein), and the oxidation of bile-acid precursors also has been suggested as being catalyzed by the d-bifunctional protein. Because of the important roles of this protein, we reinvestigated two Japanese patients previously diagnosed as having enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein) deficiency, in complementation studies. We found that both the protein and the enzyme activity of the d-bifunctional protein were hardly detectable in these patients but that the active L-bifunctional protein was present. The mRNA level in patient 1 was very low, and, for patient 2, mRNA was of a smaller size. Sequencing analysis of the cDNA revealed a 52-bp deletion in patient 1 and a 237-bp deletion in patient 2. This seems to be the first report of D-bifunctional protein deficiency. Patients previously diagnosed as cases of L-bifunctional protein deficiency probably should be reexamined for a possible d-bifunctional protein deficiency.     

Role of short-chain hydroxyacyl CoA dehydrogenases in SCHAD deficiency

Short-chain hydroxyacyl CoA dehydrogenase deficiency is an ill-defined, severe pediatric disorder of mitochondrial fatty acid β-oxidation of short-chain hydroxyacyl CoAs. To understand the relative contributions of the two known short-chain hydroxyacyl CoA dehydrogenases (HADH) tissue biopsies of six distinct family individuals were analyzed and kinetic parameters were compared. Steady-state kinetic constants for HADH 1 and HADH 2 suggest that type 1 is the major enzyme involved in mitochondrial β-oxidation of short-chain hydroxyacyl-CoAs. Two patients are heterozygous carriers of a HADH 1 polymorphism, whereas no mutation is detected in the HADH 2 gene of all patients. The data suggest that protein interactions rather than HADH mutations are responsible for the disease phenotype. Pull-down experiments of recombinant HADH 1 and 2 with human mitochondrial extracts reveal two proteins interacting with HADH 1, one of which was identified as glutamate dehydrogenase. This association provides a possible link between fatty acid metabolism and the hyperinsulinism/hyperammonia syndrome.     

Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.

Long-chain 3-hydroxyacyl-CoA dehydrogenase was extracted from the washed membrane fraction of frozen rat liver mitochondria with buffer containing detergent and then was purified. This enzyme is an oligomer with a molecular mass of 460 kDa and consisted of 4 mol of large polypeptide (79 kDa) and 4 mol of small polypeptides (51 and 49 kDa). The purified enzyme preparation was concluded to be free from the following enzymes based on marked differences in behavior of the enzyme during purification, molecular masses of the native enzyme and subunits, and immunochemical properties: enoyl-CoA hydratase, short-chain 3-hydroxyacyl-CoA dehydrogenase, peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases. The purified enzyme exhibited activities toward enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase together with the long-chain 3-hydroxyacyl-CoA dehydrogenase activity. The carbon chain length specificities of these three activities of this enzyme differed from those of the other enzymes. Therefore, it is concluded that this enzyme is not long-chain 3-hydroxyacyl-CoA dehydrogenase; rather, it is enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein.     

Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product.

The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.     

Molecular characterization of mitochondrial trifunctional protein deficiency: formation of the enzyme complex is important for stabilization of both alpha- and beta-subunits.

Mitochondrial trifunctional protein (TP) is an enzyme complex with three activities: enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. Studies on defects in this enzyme in patients with TP deficiency suggest that there are two types of defect. Patients in group 1 have normal amount of cross-reacting material by immunoblot and lack only long-chain 3-hydroxyacyl-CoA dehydrogenase activity. Patients in group 2 have a trace amount of cross-reacting material, with all three activities being low. We identified three patients in group 2, and analysis was made at the cDNA level. In patient 2, there was a heterozygous 71-bp deletion at position 110-180 in the alpha-subunit. In patients 1 and 3, there was an abnormal beta-subunit; patient 1 had an A-788-to-G substitution, and patient 3 had G-182-to-A and G-740-to-A substitutions in each of separate alleles. This is the first demonstration of disease-causing mutations in the beta-subunit. cDNA-expression experiments in patients' fibroblasts, using a vaccinia virus system, and gel filtration analysis, using patients' fibroblasts, revealed that the existence of both normal alpha- and beta-subunits, and possibly their association, are important for stabilizing TP and that A-788-to-G substitution on the beta-subunit in patient 1 seems to interfere with the association, the result being a rapid decomposition of TP.     

Peroxisomal beta-oxidation system of Candida tropicalis. Purification of a multifunctional protein possessing enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase activities

A multifunctional protein from oleate-grown cells of Candida tropicalis has been purified and partially characterized. A simple two-step purification has been developed involving ion-exchange chromatography followed by dye-ligand chromatography on blue Sepharose CL-6B. Homogeneous enzyme with a subunit Mr of 102 000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse-phase high-performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits. Three beta-oxidation activities, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase, co-purified with this protein. The ratio of the three beta-oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3-hydroxyacyl-CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein. After isopycnic centrifugation of a particulate fraction from oleate-grown cells in a linear sucrose gradient the activities of all individual beta-oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme. The relationship of this multifunctional protein to the two bifunctional beta-oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.     

Isolation and characterization of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from pig heart inner mitochondrial membrane

3-Hydroxyacyl coenzyme A (CoA) dehydrogenase-binding protein was solubilized from inner mitochondrial membrane by using taurodeoxycholate at high ionic strength. The binding protein was isolated from the suspension using 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The protein eluted from the affinity column had a molecular weight of approximately 150,000, as determined by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein is a dimer consisting of 69,000 and 71,000 molecular weight subunits. The enzyme binding capacity of this protein was tested with a polyethylene glycol precipitation method: 0.5 mg of enzyme could be precipitated together with 1 mg of binding protein, showing that 1 mol of binding protein binds 1 mol of enzyme. This protein had no affinity toward malic dehydrogenase, citrate synthase, and fumarase. The approximately 2-fold increase in the 3-hydroxyacyl-CoA dehydrogenase activity when it was measured in the presence of the binding protein is additional evidence of enzyme-binding protein interaction. When incorporated into liposomes, the binding protein retained its ability to bind 3-hydroxyacyl-CoA dehydrogenase, but did not bind malic dehydrogenase, citrate synthase, and fumarase. These results suggest that the protein isolated by us has a specific function in anchoring a beta-oxidation enzyme to the matrix surface of the mitochondrial membrane.     

Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities

Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide.     

A 5-methylcytosine hotspot responsible for the prevalent HSD17B10 mutation

Approximately half of the cases of hydroxysteroid (17β) dehydrogenase X (HSD10) deficiency are due to a missense C>T mutation in exon 4 of the HSD17B10 gene. The resulting HSD10 (p.R130C) loses most or all catalytic functions, and the males with this mutation have a much more severe clinical phenotype than those carrying p.V65A, p.L122V, or p.E249Q mutations. We found that the mutated cytosine which is + 2259 nucleotide from the ATG of the gene, is > 90% methylated in both the active and inactive X chromosomes in two normal females as well as in the X chromosome of a normal male. Since 5-methylcytosine is prone to conversion to thymine by deamination, the methylation of this cytosine in normal X chromosomes provides an explanation for the prevalence of the p.R130C mutation among patients with HSD10 deficiency. The substitution of arginine for cysteine eliminates several hydrogen bonds and reduces the van der Waals interaction between HSD10 subunits. The resulting disruption of protein structure impairs some if not all of the catalytic and non-enzymatic functions of HSD10. A meta-analysis of residual HSD10 activity in eight patients with the p.R130C mutation showed an average 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) activity of only 6 (± 5) % of the normal control level. This is significantly lower than in cells of patients with other, clinically milder mutations and suggests that the loss of HSD10/MHBD activity is a marker for the disorder.     

Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.     

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K

Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.     

The beta-oxidation system in catalase-free microbodies of the filamentous fungus Neurospora crassa. Purification of a multifunctional protein possessing 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities.

A trifunctional beta-oxidation protein, designated TFP, was purified to apparent homogeneity from oleate-induced mycelia of Neurospora crassa. 2-Enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities copurified in constant ratios with this protein when crude extracts were subjected to cation-exchange, dye-ligand, and adsorption chromatography. Trifunctionality was substantiated by coinciding enzyme activity ratios during the last two purification steps and additional chromatographic steps. The enzyme was shown to be a 365-kDa tetramer of subunits with a molecular mass of 93 kDa. Several lines of evidence suggest that these subunits are identical. Monospecific antibodies raised against the homogenous protein specifically precipitated the three enzymatic activities of TFP. Immunoblotting of fractions obtained after sucrose density gradient centrifugation of a crude extract indicated that TFP was exclusively localized in glyoxysome-like microbodies. The beta-oxidation system of N. crassa is structurally related to those of peroxisomes despite the presence of an acyl-CoA dehydrogenase rather than an acyl-CoA oxidase. A mitochondrial 2-enoyl-CoA hydratase activity was separated from TFP and purified to apparent homogeneity. The absence of all other beta-oxidation activities from mitochondria suggests that this organelle and its 2-enoyl-CoA hydratase are not involved in fatty acid degradation in N. crassa.     

Human brain short chain L-3-hydroxyacyl coenzyme A dehydrogenase is a single-domain multifunctional enzyme. Characterization of a novel 17beta-hydroxysteroid dehydrogenase.

Human brain short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was found to catalyze the oxidation of 17beta-estradiol and dihydroandrosterone as well as alcohols. Mitochondria have been demonstrated to be the proper location of this NAD+-dependent dehydrogenase in cells, although its primary structure is identical to an amyloid beta-peptide binding protein reportedly associated with the endoplasmic reticulum (ERAB). This fatty acid beta-oxidation enzyme was identified as a novel 17beta-hydroxysteroid dehydrogenase responsible for the inactivation of sex steroid hormones. The catalytic rate constant of the purified enzyme was estimated to be 0.66 min-1 with apparent Km values of 43 and 50 microM for 17beta-estradiol and NAD+, respectively. The catalytic efficiency of this enzyme for the oxidation of 17beta-estradiol was comparable with that of peroxisomal 17beta-hydroxysteroid dehydrogenase type 4. As a result, the human SCHAD gene product, a single-domain multifunctional enzyme, appears to function in two different pathways of lipid metabolism. Because the catalytic functions of human brain short chain L-3-hydroxyacyl-CoA dehydrogenase could weaken the protective effects of estrogen and generate aldehydes in neurons, it is proposed that a high concentration of this enzyme in brain is a potential risk factor for Alzheimer's disease.     

Molecular cloning of cDNA for rat mitochondrial 3-hydroxyacyl-CoA dehydrogenase

Messenger RNA for 3-hydroxyacyl-CoA dehydrogenase, a mitochondrial matrix enzyme of fatty acid beta-oxidation, was purified from livers of di(2-ethylhexyl)phthalate-treated rats by immunoadsorption of hepatic free polysomes to fixed cells of Staphylococcus aureus and enrichment for poly(A)-rich RNA by oligo(dT)-cellulose chromatography. Plasmid cDNA was constructed from this poly(A)-rich RNA by a modification of the method of Okayama and Berg and was transformed into the Escherichia coli DH1 strain. Plasmids containing cDNA sequences coding for 3-hydroxyacyl-CoA dehydrogenase were screened by differential colony hybridization, and were identified by hybrid-arrested translation and hybrid-selected translation. Plasmid pHADH-1, which contains a 1400-base-pair insert, hybridized to rat 3-hydroxyacyl-CoA dehydrogenase mRNA with a length of 1700 bases. Determination of the dehydrogenase mRNA by in vitro translation and dot-blot analysis with the cDNA probe showed that the induction of the enzyme in rat liver by di(2-ethylhexyl)phthalate could be attributed to an increase in the mRNA concentration.     

Organization of metabolic pathways in vastus lateralis of patients with chronic obstructive pulmonary disease

The objective of this study was to determine whether patients with chronic obstructive lung disease (COPD) display differences in organization of the metabolic pathways and segments involved in energy supply compared with healthy control subjects. Metabolic pathway potential, based on the measurement of the maximal activity (V(max)) of representative enzymes, was assessed in tissue extracted from the vastus lateralis in seven patients with COPD (age 67 +/- 4 yr; FEV(1)/FVC = 44 +/- 3%, where FEV(1) is forced expiratory volume in 1 s and FVC is forced vital capacity; means +/- SE) and nine healthy age-matched controls (age 68 +/- 2 yr; FEV(1)/FVC = 75 +/- 2%). Compared with control, the COPD patients displayed lower (P < 0.05) V(max) (mol.kg protein(-1).h(-1)) for cytochrome c oxidase (COX; 21.2 +/- 2.0 vs. 28.7 +/- 2.2) and 3-hydroxyacyl-CoA dehydrogenase (HADH; 2.54 +/- 0.14 vs. 3.74 +/- 0.12) but not citrate synthase (CS; 2.20 +/- 0.16 vs. 3.19 +/- 0.5). While no differences between groups were observed in V(max) for creatine phosphokinase, phosphorylase (PHOSPH), phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase, hexokinase (HEX) was elevated in COPD (P < 0.05). Enzyme activity ratios were higher (P < 0.05) for HEX/CS, HEX/COX, PHOSPH/HADH and PFK/HADH in COPD compared with control. It is concluded that COPD patients exhibit a reduced potential for both the electron transport system and fat oxidation and an increased potential for glucose phosphorylation while the potential for glycogenolysis and glycolysis remains normal. A comparison of enzyme ratios indicated greater potentials for glucose phosphorylation relative to the citric acid cycle and the electron transport chain and glycogenolysis and glycolysis relative to beta-oxidation.     

Characterization of the HSD17B4 gene: D-specific multifunctional protein 2/17beta-hydroxysteroid dehydrogenase IV.

The HSD17B4 gene codes for a 80 kDa multifunctional enzyme containing three distinct functional domains and is localized in peroxisomes. The N-terminal part exhibits 3-hydroxyacyl-CoA dehydrogenase and 17beta-hydroxysteroid dehydrogenase activity whereas the central part shows enoyl-CoA hydratase activity. The carboxy-terminal part of the protein has sterol-carrier-protein activity. The protein is widely expressed, however in several tissues like brain, uterus and lung its expression is limited to specific cells like Purkinje cells or luminal epithelium. The HSD17B4 gene consist of 24 exons and 23 introns with classical intron-exon junctions spanning more than 100 kbp. The importance of the HSD17B4 protein is stressed by the identification of patients with severe clinical abnormalities due to mutations in the HSD17B4 gene. We have now checked the consequences of one frequent mutation, G16 S, which results in inactivation of the enzyme due to loss of interaction with NAD+.