Short-chain L-3-hydroxyacyl-CoA Dehydrogenase Research Articles

Enzyme Kinetics of Native and Recombinant Secondary Alcohol Dehydrogenase originated from Micrococcus leuteus WIUJH20

A novel secondary alcohol dehydrogenase (2°‐ADH) from M. luteus WIUJH20 has been characterized and its gene cloned & expressed in this lab previously. This 2°‐ADH belongs to NAD+‐dependent short‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (SCADH) based on amino acid sequence homology. The native and recombinant 2°‐ADH have been purified to homogeneity. Both forms of the enzyme were soluble and were stable at −20°C which facilitated our kinetic studies toward various substrates. Our results showed that the 2°‐ADH has low substrate specificity: it catalyzed either the hydroxyl fatty acids or straight chain aliphatic secondary alcohols with hydroxyl group in either D or L form on a number of carbon positions; hydroxyl fatty esters were poor substrates; and cyclic aliphatic secondary alcohols and hydroxyacyl‐CoA were not substrates for both forms of 2°‐ADHs. To address the substrate specificities between the 2°‐ADH and SCADH, we have created three amino acids substitution mutants by site‐directed mutagenesis. The kinetic properties of the mutant proteins toward various substrates are compared to that of the wild‐type 2°‐ADH.This research is supported in part by Western Illinois University (WIU) Foundation and the College of Arts and Sciences.

Site‐directed mutagenesis of a novel secondary alcohol dehydrogenase from M. luteus WIUJH20

A novel secondary alcohol dehydrogenase (2o‐ADH) has been characterized in this lab previously. This 2o‐ADH is categorized as secondary alcohol dehydrogenase based on its biochemical reactions, but it belongs to NAD+‐dependent short‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (SCADH) based on amino acid sequence homology. In literatures, 2o‐ADH and SCADHs are distinguished in term of their primary structure and substrate specificities. SCADH has tight substrate specificity; it only catalyzes L‐3‐hydroxyacyl‐CoAs with various hydrocarbon chain‐length. In comparison the 2o‐ADH has low substrate specificity; it can catalyze hydroxy fatty acids with hydroxyl group in either D or L form on a number of carbon positions. However, the enzyme can not use hydroxyacyl‐CoA as a substrate. This 2o‐ADH is an excellent model system to study structure‐function relationship and determine what distinguishes their substrate specificity. We report here generation of single and double amino acid substitution mutants (D60S, A67K, and D60S + A67K) of 2o‐ADH in order to probe substrate functional group specificity.The research was supported in part by Western Illinois University Foundation.

The HADHSC Gene Encoding Short-Chain l-3-Hydroxyacyl-CoA Dehydrogenase (SCHAD) and Type 2 Diabetes Susceptibility

The short-chain l-3-hydroxyacyl-CoA dehydrogenase (SCHAD) protein is involved in the penultimate step of mitochondrial fatty acid oxidation. Previously, it has been shown that mutations in the corresponding gene (HADHSC) are associated with hyperinsulinism in infancy. The presumed function of the SCHAD enzyme in glucose-stimulated insulin secretion led us to the hypothesis that common variants in HADHSC on chromosome 4q22-26 might be associated with development of type 2 diabetes. In this study, we have performed a large-scale association study in four different cohorts from the Netherlands and Denmark (n = 7,365). Direct sequencing of HADHSC cDNA and databank analysis identified four tagging single nucleotide polymorphisms (SNPs) including one missense variant (P86L). Neither the SNPs nor haplotypes investigated were associated with the disease, enzyme function, or any relevant quantitative measure (all P > 0.1). The present study provides no evidence that the specific HADHSC variants or haplotypes examined do influence susceptibility to develop type 2 diabetes. We conclude that it is unlikely that variation in HADHSC plays a major role in the pathogenesis of type 2 diabetes in the examined cohorts.

Dominantly inherited hyperinsulinaemic hypoglycaemia

Congenital hyperinsulinism (HI), the most important cause of hypoglycaemia in early infancy, is a heterogeneous disease with two types of histological lesions, focal and diffuse, with major consequences in terms of surgical approaches. In contrast to focal islet-cell hyperplasia, always sporadic to our knowledge, diffuse hyperinsulinism is a heterogeneous disorder involving several genes, various mechanisms of pathogenic mutations and different transmissions: (i) channelopathy involving the genes encoding the sulphonylurea receptor (SUR1) or the inward-rectifying potassium channel (Kir6.2) in recessively inherited HI or more rarely dominantly inherited HI; (ii) metabolic disorders implicating the short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) enzyme inrecessively inherited HI, the glucokinase gene (GK), the glutamate dehydrogenase gene (GLUD1) when hyperammonemia is associated, dominant exercise-induced HI with still-unknown mechanism, and more recently the human insulin receptor gene in dominantly inherited hyperinsulinism. Thus, dominant HI disorders always correspond to diffuse HI, where most hypoglycaemia occur in infancy, and are sensitive to medical treatment. Channel causes could be due to dominant negative mutation with one abnormality in channels composed of four Kir6.2 subunits and four SUR1 subunits, leading to a complete destruction of the channel structure or function, or due to haploinsufficiency with only one functional allele, leading to 50% of functional protein, which is not sufficient to obtain enough opened channels to maintain the membrane depolarized. Metabolic causes are due to a gain of function of enzyme activity (deregulated enzymes), except for physical exercise-induced hyperinsulinaemic hypoglycaemia, of still-unknown cause. Congenital hyperinsulinism (HI) is the most important cause of hypoglycaemia in early infancy (Aynsley-Green et al 2000; Cornblath et al 1990; Pagliara et al 1973; Thomas et al 1977). The inappropriate oversecretion of insulin is responsible for profound hypoglycaemia that requires aggressive treatment to prevent severe and irreversible brain damage (Volpe 1995). HI is a heterogeneous disease associated with several genes, various mechanisms of pathogenic mutations and different transmissions (Dunne et al 2004).

Presence of hydroxysteroid dehydrogenase type 10 in amyloid plaques (APs) of Hsiao’s APP-Sw transgenic mouse brains, but absence in APs of Alzheimer’s disease brains

This immunocytochemical study using two anti-amyloid β-protein (Aβ) monoclonal antibodies, 4G8 and 6E10, revealed the presence of Aβ in both amyloid plaques (APs) and blood vessels of brains of Hsiao’s APP-Sw transgenic mice (also known as Tg2576) and human Alzheimer’s disease (AD) brains. Further study using both monoclonal (5F3) and polyclonal (R-228) antibodies to hydroxysteroid dehydrogenase type 10 (HSD-10) [formerly called SCHAD (short-chain l-3-hydroxyacyl-CoA dehydrogenase); also called ERAB (endoplasmic-reticulum-associated amyloid beta-peptide-binding protein)] indicated that HSD-10 was present in the APs of Tg2576 mice but was absent or immunocytochemically undetectable in the APs of AD brains. Our observations also revealed that HSD-10 was present in the blood vessels of both Tg2576 mice and AD brains. Immunogold electron microscopy also indicated that HSD-10 was present in the amyloid fibers (AFs), mitochondria, nuclear heterochromatin, and nucleolus of Tg2576 mouse brains but was absent in APs of AD brains. These results suggest that the human APP gene transferred to mice may induce overexpression of HSD-10 in mouse APs and in various other cellular components of mouse brains. It is also possible that the human APP gene responsible for HSD-10 deposition in APs of these Tg2576 mice brains is different from that of AD brains. Alternatively, the HSD-10 gene and APP gene may function independently in AD brains. Despite these differences, the Tg2576 mouse, as shown in this study, is a proper animal model for the study of AD and also for the investigation of HSD-10.

Hyperinsulinism in short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency reveals the importance of β-oxidation in insulin secretion

A female infant of nonconsanguineous Indian parents presented at 4 months with a hypoglycemic convulsion. Further episodes of hypoketotic hypoglycemia were associated with inappropriately elevated plasma insulin concentrations. However, unlike other children with hyperinsulinism, this patient had a persistently elevated blood spot hydroxybutyrylcarnitine concentration when fed, as well as when fasted. Measurement of the activity of L-3-hydroxyacyl-CoA dehydrogenase in cultured skin fibroblasts with acetoacetyl-CoA substrate showed reduced activity. In fibroblast mitochondria, the activity was less than 5% that of controls. Sequencing of the short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) genomic DNA from the fibroblasts showed a homozygous mutation (C773T) changing proline to leucine at amino acid 258. Analysis of blood from the parents showed they were heterozygous for this mutation. Western blot studies showed undetectable levels of immunoreactive SCHAD protein in the child’s fibroblasts. Expression studies showed that the P258L enzyme had no catalytic activity. We conclude that C773T is a disease-causing SCHAD mutation. This is the first defect in fatty acid β-oxidation that has been associated with hyperinsulinism and raises interesting questions about the ways in which changes in fatty acid and ketone body metabolism modulate insulin secretion by the β cell. The patient’s hyperinsulinism was easily controlled with diazoxide and chlorothiazide.

A new software routine that automates the fitting of protein X-ray crystallographic electron-density maps.

The classical approach to building the amino-acid residues into the initial electron-density map requires days to weeks of a skilled investigator's time. Automating this procedure should not only save time, but has the potential to provide a more accurate starting model for input to refinement programs. The new software routine MAID builds the protein structure into the electron-density map in a series of sequential steps. The first step is the fitting of the secondary alpha-helix and beta-sheet structures. These 'fits' are then used to determine the local amino-acid sequence assignment. These assigned fits are then extended through the loop regions and fused with the neighboring sheet or helix. The program was tested on the unaveraged 2.5 A selenomethionine multiple-wavelength anomalous dispersion (SMAD) electron-density map that was originally used to solve the structure of the 291-residue protein human heart short-chain L-3-hydroxyacyl-CoA dehydrogenase (SHAD). Inputting just the map density and the amino-acid sequence, MAID fitted 80% of the residues with an r.m.s.d. error of 0.43 A for the main-chain atoms and 1.0 A for all atoms without any user intervention. When tested on a higher quality 1.9 A SMAD map, MAID correctly fitted 100% (418) of the residues. A major advantage of the MAID fitting procedure is that it maintains ideal bond lengths and angles and constrains phi/psi angles to the appropriate Ramachandran regions. Recycling the output of this new routine through a partial structure-refinement program may have the potential to completely automate the fitting of electron-density maps.

Molecular mechanism of mitochondrial short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) involved in Alzheimer's disease

Molecular mechanism of mitochondrial short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) involved in Alzheimer's disease

Function of human brain short chain L-3-hydroxyacyl coenzyme A dehydrogenase in androgen metabolism

Human brain short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) has been demonstrated to be a unique 3α-hydroxysteroid dehydrogenase (HSD) that can convert 5α-androstane-3α,17β-diol (3α-adiol) to dihydrotestosterone (DHT), whose affinity to the androgen receptor is 10 5-fold higher than that of 3α-adiol. The catalytic efficiency of human SCHAD for this oxidative 3α-HSD reaction was estimated to be 164 min −1 mM −1, about 10-fold higher than that measured for the backward reaction. Thus, human brain SCHAD may function in androgen metabolism as a new kind of 3α-HSD by counteracting all other known 3α-HSDs, which would unidirectionally catalyze the reduction of DHT to the almost inactive 3α-adiol. Human SCHAD is identical to an amyloid-β binding protein (ERAB) involved in Alzheimer’s disease, which was previously reported to be associated with the endoplasmic reticulum. This protein is, in fact, localized in mitochondria, not endoplasmic reticulum, as evidenced by immunocytochemical studies and its noncleavable mitochondrial targeting sequence and lack of endoplasmic reticulum targeting signals or transmembrane segments. These results prompt the suggestion that the mitochondrion plays not only an essential role in the initial step of steroidogenesis, but also important roles in the intracellular homeostasis of sex steroid hormones. Northern blot analysis revealed that the human SCHAD gene is expressed in both gonadal and peripheral tissues including the prostate whose growth notably requires DHT, the most potent androgen. This study represents the first report of a 3α-HSD that could act to generate DHT from 3α-adiol and thereby maintain intracellular DHT levels. We propose that inhibitors of the 3α-HSD activity of human brain SCHAD could be useful for the treatment of benign prostatic hyperplasia and other disorders involving DHT metabolism, in combination with known inhibitors of steroid 5α-reductases.

Intrinsic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities of human mitochondrial short-chain L-3-hydroxyacyl-CoA dehydrogenase

The alcohol dehydrogenase (ADH) activity of human short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) has been characterized kinetically. The kcat of the purified enzyme was estimated to be 2.2 min-1, with apparent Km values of 280 mM and 22μM for 2-propanol and NAD+, respectively. The kcat of the ADH activity was three orders of magnitude less than the L-3-hydroxyacyl-CoA dehydrogenase activity but was comparable with that of the enzyme's hydroxysteroid dehydrogenase (HSD) activity for oxidizing 17β-oestradiol [He, Merz, Mehta, Schulz and Yang (1999) J. Biol. Chem. 274, 15014-15019]. However, the kcat values of intrinsic ADH and HSD activities of human SCHAD were found to be two orders of magnitude less than those reported for endoplasmic-reticulum-associated amyloid β-peptide-binding protein (ERAB) [Yan, Shi, Zhu, Fu, Zhu, Zhu, Gibson, Stern, Collison, Al-Mohanna et al. (1999) J. Biol. Chem. 274, 2145-2156]. Since human SCHAD and ERAB apparently possess identical amino acid sequences, their catalytic properties should be identical. The recombinant SCHAD has been confirmed to be the right gene product and not a mutant variant. Steady-state kinetic measurements and quantitative analyses reveal that assay conditions such as pH and concentrations of coenzyme and substrate do not account for the kinetic differences reported for ERAB and SCHAD. Rather problematic experimental procedures appear to be responsible for the unrealistically high catalytic rate constants of ERAB. Eliminating the confusion surrounding the catalytic properties of this important multifunctional enzyme paves the way for exploring its role(s) in the pathogenesis of Alzheimer's disease.

Intrinsic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities of human mitochondrial short-chain L-3-hydroxyacyl-CoA dehydrogenase

The alcohol dehydrogenase (ADH) activity of human short-chain l-3-hydroxyacyl-CoA dehydrogenase (SCHAD) has been characterized kinetically. The k(cat) of the purified enzyme was estimated to be 2. 2 min(-1), with apparent K(m) values of 280 mM and 22microM for 2-propanol and NAD(+), respectively. The k(cat) of the ADH activity was three orders of magnitude less than the l-3-hydroxyacyl-CoA dehydrogenase activity but was comparable with that of the enzyme's hydroxysteroid dehydrogenase (HSD) activity for oxidizing 17beta-oestradiol [He, Merz, Mehta, Schulz and Yang (1999) J. Biol. Chem. 274, 15014-15019]. However, the k(cat) values of intrinsic ADH and HSD activities of human SCHAD were found to be two orders of magnitude less than those reported for endoplasmic-reticulum-associated amyloid beta-peptide-binding protein (ERAB) [Yan, Shi, Zhu, Fu, Zhu, Zhu, Gibson, Stern, Collison, Al-Mohanna et al. (1999) J. Biol. Chem. 274, 2145-2156]. Since human SCHAD and ERAB apparently possess identical amino acid sequences, their catalytic properties should be identical. The recombinant SCHAD has been confirmed to be the right gene product and not a mutant variant. Steady-state kinetic measurements and quantitative analyses reveal that assay conditions such as pH and concentrations of coenzyme and substrate do not account for the kinetic differences reported for ERAB and SCHAD. Rather problematic experimental procedures appear to be responsible for the unrealistically high catalytic rate constants of ERAB. Eliminating the confusion surrounding the catalytic properties of this important multifunctional enzyme paves the way for exploring its role(s) in the pathogenesis of Alzheimer's disease.

Human Brain Short Chain l-3-Hydroxyacyl Coenzyme A Dehydrogenase Is a Single-domain Multifunctional Enzyme: CHARACTERIZATION OF A NOVEL 17β-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.

Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism.

Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the beta-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other alpha/beta dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central beta-sheet. The second, or C-terminal, domain is primarily alpha-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.

Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: sequence analysis and crystal structure determination.

Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.

Human Short-Chain L-3-Hydroxyacyl-CoA Dehydrogenase: Cloning and Characterization of the Coding Sequence

The cDNA encompassing the complete coding sequence of human liver short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was isolated and characterized. Screening of a cDNA library combined with rapid amplification of 5′ cDNA ends resulted in a SCHAD cDNA sequence of 1877 bp. It encodes a protein of 314 amino acids with a calculated molecular weight of 34,3 kDa containing a mitochondrial import signal peptide of 12 amino acids and 302 amino acids of mature SCHAD protein. The deduced amino acid sequence of the mature protein shows a 92 percent identity with SCHAD from pig heart. Northern blot analysis reveals SCHAD mRNA to be expressed in liver, kidney, pancreas, heart and skeletal muscle. The human SCHAD gene was mapped by fluorescence in situ hybridization to chromosome 4q22-26.

Metabolic myopathies

Disorders of glycogen, lipid or mitochondrial metabolism may cause two main clinical syndromes, namely (1) progressive weakness (eg, acid maltase, debrancher enzyme, and brancher enzyme deficiencies among the glycogenoses; long- and very-long-chain acyl-CoA dehydrogenase (LCAD, VLCAD), and trifunctional enzyme deficiencies among the fatty acid oxidation (FAO) defects; and mitochondrial enzyme deficiencies) or (2) acute, recurrent, reversible muscle dysfunction with exercise intolerance and acute muscle breakdown or myoglobinuria (with or without cramps) (eg, phosphorylase (PPL), phosphorylase b kinase (PBK), phosphofructokinase (PFK), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGAM), and lactate dehydrogenase (LDH) among the glycogenoses and carnitine palmitoyltransferase II (CPT II) deficiency among the disorders of FAO or (3) both (eg, PPL, PBK, PFK among the glycogenoses; LCAD, VLCAD, short-chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), and trifunctional enzyme deficiencies among the FAO defects; and multiple mitochondrial DNA (mtDNA) deletions). Myoadenylate deaminase deficiency, a purine nucleotide cycle defect, is somewhat controversial and is characterized by exercise-related cramps leading rarely to myoglobinuria.