Song-Yu Yang

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 chainl-3-hydroxyacyl-CoA dehydrogenase (SCHAD) was found to catalyze the oxidation of 17β-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 β-peptide binding protein reportedly associated with the endoplasmic reticulum (ERAB). This fatty acid β-oxidation enzyme was identified as a novel 17β-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 K m values of 43 and 50 μmfor 17β-estradiol and NAD+, respectively. The catalytic efficiency of this enzyme for the oxidation of 17β-estradiol was comparable with that of peroxisomal 17β-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.

A HUMAN BRAIN L-3-HYDROXYACYL-COENZYME A DEHYDROGENASE IS IDENTICAL TO AN AMYLOID BETA -PEPTIDE-BINDING PROTEIN INVOLVED IN ALZHEIMER'S DISEASE

A novel l-3-hydroxyacyl-CoA dehydrogenase from human brain has been cloned, expressed, purified, and characterized. This enzyme is a homotetramer with a molecular mass of 108 kDa. Its subunit consists of 261 amino acid residues and has structural features characteristic of short chain dehydrogenases. It was found that the amino acid sequence of this human brain enzyme is identical to that of an endoplasmic reticulum amyloid β-peptide-binding protein (ERAB), which mediates neurotoxicity in Alzheimer’s disease (Yan, S. D., Fu, J., Soto, C., Chen, X., Zhu, H., Al-Mohanna, F., Collison, K., Zhu, A., Stern, E., Saido, T., Tohyama, M., Ogawa, S., Roher, A., and Stern, D. (1997) Nature 389, 689–695). The purification of human brain short chainl-3-hydroxyacyl-CoA dehydrogenase made it possible to characterize the structural and catalytic properties of ERAB. This NAD+-dependent dehydrogenase catalyzes the reversible oxidation of l-3-hydroxyacyl-CoAs to form 3-ketoacyl-CoAs, but it does not act on the d-isomers. The catalytic rate constant of the purified enzyme was estimated to be 37 s−1 with apparent K m values of 89 and 20 μm for acetoacetyl-CoA and NADH, respectively. The activity ratio of this enzyme for substrates with chain lengths of C4, C8, and C16 was ∼1:2:2. The human short chain l-3-hydroxyacyl-CoA dehydrogenase gene is organized into six exons and five introns and maps to chromosome Xp11.2. The amino-terminal NAD-binding region of the dehydrogenase is encoded by the first three exons, whereas the other exons code for the carboxyl-terminal substrate-binding region harboring putative catalytic residues. The results of this study lead to the conclusion that ERAB involved in neuronal dysfunction is encoded by the human short chainl-3-hydroxyacyl-CoA dehydrogenase gene.

Multiple functions of type 10 17β-hydroxysteroid dehydrogenase

Human 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a mitochondrial enzyme encoded by the SCHAD gene, which escapes chromosome X inactivation. 17β-HSD10/SCHAD mutations cause a spectrum of clinical conditions, from mild mental retardation to progressive infantile neurodegeneration. 17β-HSD10/SCHAD is essential for the metabolism of isoleucine and branched-chain fatty acids. It can inactivate 17β-estradiol and steroid modulators of GABA A receptors, and convert 5α-androstanediol into 5α-dihydrotestosterone (DHT). Certain malignant prostatic epithelial cells contain high levels of 17β-HSD10, generating 5α-DHT in the absence of testosterone. 17β-HSD10 has an affinity for amyloid-β peptide, and might be linked to the mitochondrial dysfunction seen in Alzheimers disease. This versatile enzyme might provide a new drug target for neuronal excitability control and for intervention in Alzheimers disease and certain cancers.

3-Hydroxyacyl-CoA dehydrogenase and short chain 3-hydroxyacyl-CoA dehydrogenase in human health and disease

3‐Hydroxyacyl‐CoA dehydrogenase (HAD) functions in mitochondrial fatty acid β‐oxidation by catalyzing the oxidation of straight chain 3‐hydroxyacyl‐CoAs. HAD has a preference for medium chain substrates, whereas short chain 3‐hydroxyacyl‐CoA dehydrogenase (SCHAD) acts on a wide spectrum of substrates, including steroids, cholic acids, and fatty acids, with a preference for short chain methyl‐branched acyl‐CoAs. Therefore, HAD should not be referred to as SCHAD. SCHAD is not a member of the HAD family, but instead, belongs to the short chain dehydrogenase/reductase superfamily. Previously reported cases of SCHAD deficiency are due to an inherited HAD deficiency. SCHAD, also known as 17β‐hydroxysteroid dehydrogenase type 10, is important in brain development and aging. Abnormal levels of SCHAD in certain brain regions may contribute to the pathogenesis of some neural disorders. The human SCHAD gene and its protein product, SCHAD, are potential targets for intervention in conditions, such as Alzheimers disease, Parkinsons disease, and an X‐linked mental retardation, that may arise from the impaired degradation of branched chain fatty acid and isoleucine.

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 3alpha-hydroxysteroid dehydrogenase (HSD) that can convert 5alpha-androstane-3alpha, 17beta-diol (3alpha-adiol) to dihydrotestosterone (DHT), whose affinity to the androgen receptor is 10(5)-fold higher than that of 3alpha-adiol. The catalytic efficiency of human SCHAD for this oxidative 3alpha-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 3alpha-HSD by counteracting all other known 3alpha-HSDs, which would unidirectionally catalyze the reduction of DHT to the almost inactive 3alpha-adiol. Human SCHAD is identical to an amyloid-beta binding protein (ERAB) involved in Alzheimers 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 3alpha-HSD that could act to generate DHT from 3alpha-adiol and thereby maintain intracellular DHT levels. We propose that inhibitors of the 3alpha-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 5alpha-reductases.

Type 10 17beta-hydroxysteroid dehydrogenase catalyzing the oxidation of steroid modulators of γ-aminobutyric acid type A receptors

The steroids allopregnanolone and allotetrahydrodeoxycorticosterone (3alpha,5alpha-THDOC) are positive allosteric modulators of GABA(A) receptors, generated by the reduction of 5alpha-dihydroprogesterone (5alpha-DHP) and 5alpha-DHDOC, respectively, under the catalysis of human type 3 3alpha-hydroxysteroid dehydrogenase (HSD). However, brain enzymes catalyzing the conversion of such tetrahydrosteroids back to the corresponding 5alpha-dihydrosteroids remain to be identified. Characterization of human type 10 17beta-HSD provides a new insight into its importance for the oxidation of steroid modulators of GABA(A) receptors. The apparent catalytic efficiency (k(cat)/K(m)) of this enzyme for the oxidation of allopregnanolone and 3alpha,5alpha-THDOC are 432 and 1381 min(-1) mM(-1), respectively. This enzyme has negligible 3-ketosteroid reductase activity for 5alpha-DHP and 5alpha-DHDOC even in an acidic environment. Immunoreactivity against 17beta-HSD10 was found in a number of neuronal populations. Taken together, evidence suggests that 17beta-HSD10 is the brain enzyme capable of catalyzing the oxidation of steroid modulators of GABA(A) receptors.

Oxidative 3α-hydroxysteroid dehydrogenase activity of human type 10 17β-hydroxysteroid dehydrogenase

Abstract In vitro enzyme assays have demonstrated that human type 10 17β-hydroxysteroid dehydrogenase (17β-HSD10) catalyzes the oxidation of 5α-androstane-3α,17β-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17β-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17β-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17β-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17β-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3α-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17β-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.

Assay of l-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.

Inhibition of enoyl-CoA hydratase by long-chain L-3-hydroxyacyl-CoA and its possible effect on fatty acid oxidation.

The kinetics of bovine liver enoyl-CoA hydratase (EC 4.2.1.17) or crotonase with 2-trans-hexadecenoyl-CoA as a substrate were studied because different rates were obtained with two assay methods based on measurements of substrate utilization and product formation, respectively. L-3-Hydroxyhexadecanoyl-CoA, the product of the crotonase-catalyzed hydration of 2-trans-hexadecenoyl-CoA, was found to be a strong competitive inhibitor of the enzyme with a Ki of 0.35 microM. In contrast the short-chain product, L-3-hydroxybutyryl-CoA, is a weak competitive inhibitor with a Ki of 37 microM. L-3-Hydroxyhexadecanoyl-CoA is a much stronger inhibitor of crotonase than are other short-chain and long-chain intermediates of beta-oxidation and crotonase is more severely inhibited by this compound than are all beta-oxidation enzymes tested so far. Determination of true kinetic parameters for the crotonase-catalyzed hydration of long-chain substrates requires the removal of product in a coupled assay. When this was done, the Km for 2-trans-hexadecenoyl-CoA with bovine liver crotonase was found to be only 9 microM. It is suggested that under conditions of restricted beta-oxidation, when 3-hydroxyacyl-CoAs accumulate in mitochondria, the inhibition of crotonase by long-chain 3-hydroxyacyl-CoAs may limit the further degradation of medium-chain and short-chain intermediates of beta-oxidation.

Molecular cloning, modeling, and localization of rat type 10 17β-hydroxysteroid dehydrogenase

Rat and mouse complementary DNAs of type 10 17beta-hydroxysteroid dehydrogenase were cloned and sequenced. The mouse cDNA clones sequence corrected the previously published nucleotide and amino acid sequence of mouse endoplasmic reticulum-associated beta-amyloid-binding protein. A subunit of the rat enzyme consists of 261 amino acid residues with a calculated molecular mass of 27250 Da. Compared with its human counterpart, rat 17betaHSD type 10 shows 88% identity. Mouse 17betaHSD type 10 is composed of 261 amino acid residues with a calculated molecular mass of 27274 Da. There is 95% identity between the two rodent enzymes. A stereostructure model of rat 17betaHSD type 10 was constructed based on its amino acid sequence. Similar to human type 10 17betaHSD, the rodent enzymes also displayed relatively higher 3alphaHSD activity than 17betaHSD activity. Intracellular localization of rat 17betaHSD type 10 has been determined by subcellular fractionation and confocal microscopy studies. The results unequivocally establish that this is a nuclear gene-encoded mitochondrial enzyme, and that this 17betaHSD is not associated with the endoplasmic reticulum. The unique location distinguishes type 10 from other types of 17beta-hydroxysteroid dehydrogenases.