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The tissue distribution, subcellular localization, and metabolic functions of human 17beta-hydroxysteroid dehydrogenase type 10/short chain L-3-hydroxyacyl-CoA dehydrogenase have been investigated. Human liver and gonads are abundant in this enzyme, but it is present in only negligible amounts in skeletal muscle. Its N-terminal sequence is a mitochondrial targeting sequence, but is not required for directing this protein to mitochondria. Immunocytochemical studies demonstrate that this protein, which has been referred to as ER-associated amyloid beta-binding protein (ERAB), is not detectable in the ER of normal tissues. We have established that protocols employed to investigate the subcellular distribution of ERAB yield ER fractions rich in mitochondria. Mitochondria-associated membrane fractions believed to be ER fractions were employed in ERAB/Abeta-binding alcohol dehydrogenase studies. The present studies establish that in normal tissues this protein is located in mitochondria. This feature distinguishes it from all known 17beta-hydroxysteroid dehydrogenases, and endows mitochondria with the capability of modulating intracellular levels of the active forms of sex steroids.

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 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 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.

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-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.

Rat heart and liver cDNAs for precursor of L-3-hydroxyacyl-CoA dehydrogenase have been cloned and sequenced. The results indicate that these different rat organs express identical dehydrogenases. Furthermore, pig heart mRNA for L-3-hydroxyacyl-CoA dehydrogenase precursor was amplified by reverse transcription-polymerase chain reaction, and all the cDNA clones were found to encode a precursor of liver L-3-hydroxyacyl-CoA dehydrogenase (X.-Y. He, S.-Y. Yang, Biochim. Biophys. Acta 1392 (1998) 119-126) but not the well-documented heart form of the dehydrogenase (K.G. Bitar et al., FEBS Lett. 116 (1980) 196-198). Sequencing data and other evidence establish that the pig, like the rat, has the same dehydrogenase in heart and liver. Since the size and structure of pig heart L-3-hydroxyacyl-CoA dehydrogenase are identical to the pig liver dehydrogenase, reports that relied on the published sequence of the pig heart dehydrogenase need to be re-evaluated. For example, the signature pattern of the L-3-hydroxyacyl-CoA dehydrogenase family is HXFXPX3MXLXE. Furthermore, the published crystal structure of the pig heart dehydrogenase that substantiated each subunit comprising 307 residues with a mercury-binding residue at position 204 (J.J. Birktoft et al., Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 8262-8266) must be re-examined in accordance with this revelation.

A novel L-3-hydroxyacyl-CoA dehydrogenase from pig liver has been cloned, expressed, purified, and characterized. This enzyme is a homodimer with a molecular mass of 65.6 kDa, and is distinguished from the dehydrogenase of pig heart by its structural features and catalytic properties. Its subunit, consisting of 302 amino acid residues, has two additional residues in a key region of the active center while it lacks a sequence of seven residues in the NAD+-binding domain, when compared with the subunit of pig heart enzyme. In addition, there are substitutions of four single residues. The catalytic efficiency of pig liver dehydrogenase was significantly greater than that of the heart enzyme for short-chain substrate, but its catalytic rates declined with an increase in substrate chain-lengths. The distinction between pig liver and heart dehydrogenases cannot be attributed to a species difference, and thus it is concluded that there exist different isoforms of monofunctional L-3-hydroxyacyl-CoA dehydrogenases in pig. High level expression of mitochondrial L-3-hydroxyacyl-CoA dehydrogenase in Escherichia coli has provided a very convenient way to purify this important beta-oxidation enzyme. There is substantial homology between pig liver dehydrogenase and various multifunctional beta-oxidation enzymes in the active center of these enzymes; a consensus sequence, HX3PX1-3MXLXE, is proposed as the signature sequence of l-3-hydroxyacyl-CoA dehydrogenases., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

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 beta-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 chain L-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 Km values of 89 and 20 microM for acetoacetyl-CoA and NADH, respectively. The activity ratio of this enzyme for substrates with chain lengths of C4, C8, and C16 was approximately 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 chain L-3-hydroxyacyl-CoA dehydrogenase gene.

His450 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase [He, X-Y., & Yang, S.-Y. (1996) Biochemistry 35, 9625-9630]. To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine. The resulting mutant complexes were overproduced and characterized. Their component enzymes other than the dehydrogenase were affected very slightly. Removal of the beta-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited. In contrast, the alpha/Glu462-->Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the k(m) for the substrate. The catalytic properties of the alpha/Glu462-->Gln mutant complex were found to be similar to those of the alpha/Glu462-->Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher. It is concluded that the negative charge of the gamma-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase. The pKa of His450 in the E.NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln. It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered. Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.

Multienzyme complexes of fatty acid oxidation from Escherichia coli with Gln or Ala substituting for His450 or with Ala in place of Gly322 in the large alpha-subunit have been purified and characterized. The alpha/Gly322-->Ala mutation did not significantly affect the catalytic efficiencies (kcat/k(m)) of different component enzymes except for a 6.1-fold decrease in the kcat/k(m) of L-3-hydroxyacyl-CoA dehydrogenase and a 10-fold increase in the k(m) for NADH. This observation confirms the prediction [Yang, X.-Y. H., Schulz, H., Elzinga, M., & Yang, S.-Y. (1991) Biochemistry 30, 6788-6795] that the E. coli dehydrogenase has an NAD-binding site at its amino-terminal domain and structurally resembles the pig heart dehydrogenase. The pH dependence of the kcat/k(m) of the E. coli dehydrogenase suggested the catalytic involvement of an amino acid residue with a pKa of 6, which is presumably a histidine residue as proposed previously on the basis of chemical modifications. Since His450 of the E. coli multifunctional protein is the only histidine conserved in all known L-3-hydroxyacyl-CoA dehydrogenases, and since its counterpart in pig heart enzyme appeared to be close to the 3-keto group of the fatty acyl moiety of the substrate, His450 was replaced by either Gln or Ala. The catalytic properties of 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and delta 3-cis-delta 2-trans-enoyl-CoA isomerase of the alpha/His450-->Gln mutant complex were virtually unchanged except for a small decrease in the kcat values of the latter two enzymes. In contrast, the dehydrogenase of this mutant complex was almost inactive due to a greater than 3000-fold decrease in its kcat and a 6-fold increase in the k(m) for NADH. The alpha/His450-->Ala mutant complex showed similar catalytic behaviors. Taken together, several lines of evidence lead to the conclusion that His450 is the catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase of the E. coli multifunctional fatty acid oxidation protein.

Multienzyme complexes of fatty acid oxidation from Escherichia coli with either an alpha/Glu139-->Gln or an alpha/Arg134-->Gln mutation in the large alpha-subunit have been overproduced and characterized. The catalytic properties of the five different component enzymes of the alpha/Arg134-->Gln mutant complex showed no significant changes as compared with those of the wild type complex. In contrast, the 3-hydroxyacyl-coenzyme A (CoA) epimerase activity of the alpha/Glu139-->Gln mutant complex was not detected, and this mutant complex has lost almost all of the enoyl-CoA hydratase activity due to a greater than 3000-fold decrease in the kcat of the enoyl-CoA hydratase without a significant change in the Km value. The catalytic properties of 3-ketoacyl-CoA thiolase and L-3-hydroxyacyl-CoA dehydrogenase were virtually unaffected by the mutation. Together, these observations lead to the conclusion that the gamma-carboxylic group of Glu139 functions as a catalytic base in the dehydration of both D- and L-3-hydroxyacyl-CoA. These findings also support a dehydration/hydration mechanism for 3-hydroxyacyl-CoA epimerase but do not agree with an epimerase activity independent of enoyl-CoA hydratase as proposed for the glyoxysomal tetrafunctional protein [Preisig-Müller, R., Gühnemann-Schäfer, K., & Kindl, H. (1994) J. Biol. Chem. 269, 20475-20481]. Since this mutation caused the kcat of delta 3-cis-delta 2-trans-enoyl-CoA isomerase to decrease by only 60%, even though the Km value was significantly increased, it seems that Glu139 of the E. coli multifunctional protein does not function as a catalytic residue in the isomerization reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of a trifunctional beta-oxidation complex in pig heart and its relationship to the known long-chain enoyl-CoA hydratase (EC 4.2.1.74) from pig heart mitochondria were investigated. For this study, the complex was partially purified by chromatography on DEAE-cellulose in the absence of detergents and was purified to near homogeneity in the presence of Triton X-100. Both enzyme preparations contained long-chain specific activities of enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase but were virtually inactive toward short-chain (C4) substrates. Both preparations exhibited very low or no activities of delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) and 2,4-dienoyl-CoA reductase (EC 1.3.1.34). The molecular weights of the two subunits of the pig heart complex were estimated to be 81,000 and 45,000, respectively. The partially purified preparation, obtained in the absence of detergent, was identified as a membranous fraction enriched with respect to the inner mitochondrial membrane. It is concluded that long-chain enoyl-CoA hydratase is a component enzyme of the trifunctional beta-oxidation complex which is associated with the inner membrane of pig heart mitochondria.

Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392. The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon. Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes. Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased. These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide. The results of this study, together with published data (S.-Y. Yang and H. Schulz, J. Biol. Chem. 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene.

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.