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A Human Brain l-3-Hydroxyacyl-coenzyme A Dehydrogenase Is Identical to an Amyloid β-Peptide-binding Protein Involved in Alzheimer's Disease

1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês

10.1074/jbc.273.17.10741

1083-351X

Xue‐Ying He, Horst Schulz, Song‐Yu Yang,

Amino Acid Enzymes and Metabolism

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. 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. l-3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) catalyzes the third step of the fatty acid β-oxidation pathway:l-3-hydroxyacyl-CoA + NAD+ ⇄ 3-ketoacyl-CoA + NADH + H+ (1Schulz H. Biochim. Biophys. Acta. 1991; 1081: 109-120Crossref PubMed Scopus (307) Google Scholar). This reaction is known to be catalyzed by mitochondrial monofunctional l-3-hydroxyacyl-CoA dehydrogenase and by prokaryotic and eukaryotic multifunctional β-oxidation enzymes that possess an l-3-hydroxyacyl-CoA dehydrogenase functional domain (2Yang X.-Y.H. Schulz H. Elzinga M. Yang S.-Y. Biochemistry. 1991; 30: 6788-6795Crossref PubMed Scopus (54) Google Scholar, 3Yang S.-Y. Comp. Biochem. Physiol. B Comp. Biochem. 1994; 109: 557-566Crossref PubMed Scopus (15) Google Scholar). The catalytic residue of this kind of dehydrogenase was recently identified to be a conserved histidine (4He X.-Y. Yang S.-Y. Biochemistry. 1996; 35: 9625-9630Crossref PubMed Scopus (28) Google Scholar), and a conserved glutamate residue is also required for high catalytic efficiency (5He X.-Y. Deng H. Yang S.-Y. Biochemistry. 1997; 36: 261-268Crossref PubMed Scopus (20) Google Scholar). The catalytic residue ofl-3-hydroxyacyl-CoA dehydrogenase was proposed to interact with the conserved glutamate, and this electrostatic interaction seemed to be strengthened by the binding of substrate (5He X.-Y. Deng H. Yang S.-Y. Biochemistry. 1997; 36: 261-268Crossref PubMed Scopus (20) Google Scholar). However, we were surprised to see that a catalytic His-Glu pair is not present in the newly isolated bovine liver type II dehydrogenase (6Kobayashi A. Jian L.L. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar, 7Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar), which is not homologous to any of the known l-3-hydroxyacyl-CoA dehydrogenases. More interestingly, it was reported that this new type of l-3-hydroxyacyl-CoA dehydrogenase was not found in human liver by either Northern blot or immunoblot analysis (6Kobayashi A. Jian L.L. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar, 7Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar). As a result, several important questions emerged. Do humans have anl-3-hydroxyacyl-CoA dehydrogenase of this kind? If such a dehydrogenase exists in some human organ(s), what are its possible roles in human cells? Where is the human dehydrogenase gene located, and how is the gene organized? To find answers to these questions, we have cloned the cDNA encoding human brain short chainl-3-hydroxyacyl-CoA dehydrogenase 1This enzyme, which usesl-3-hydroxyacyl-CoA substrates of various chain lengths, is a new member of the short chain dehydrogenase family. and then mapped its gene and analyzed the gene structure as well. Moreover, the enzyme of interest was purified so that the structural and catalytic properties of this human brain dehydrogenase could be successfully characterized. During the preparation of this manuscript, a 262-amino acid ERAB 2The abbreviations used are: ERAB, endoplasmic reticulum amyloid β-peptide-binding protein; PCR, polymerase chain reaction; IPTG, isopropyl-β-d-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s); RACE, rapid amplification of cDNA ends. protein detected by use of the yeast two-hybrid system to screen human brain and HeLa cell cDNA libraries was reported to bind amyloid β-peptide (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). A fusion protein, ERAB with a polyhistidine tag (His-ERAB), was generated and used to study the interaction of ERAB with amyloid β-peptide, and this protein was shown to mediate the cellular toxicity of amyloid β-peptide. ERAB, an important factor contributing to the neuronal dysfunction of Alzheimer's disease, was proposed to be a hydroxysteroid dehydrogenase (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). In this paper, we report the structure and organization of the human short chain l-3-hydroxyacyl-CoA dehydrogenase gene and the structural and catalytic properties of its gene product. The primary structure of human brain l-3-hydroxyacylCoA dehydrogenase was found to be identical to that of ERAB, and thus, ERAB proved to be an l-3-hydroxyacyl-CoA dehydrogenase. NAD+, NADH, CoASH, sodiumdl-3-hydroxybutyrate, pig heartl-3-hydroxyacyl-CoA dehydrogenase, and all other standard biochemicals were obtained from Sigma. 2-Octynoic acid was purchased from Aldrich. d-3-Hydroxybutyryl-CoA was provided by Metabolix Co. Acetoacetyl-CoA (9Seubert W. Biochem. Prep. 1960; 7: 80-83Google Scholar) and 2-hexadecynoic acid (10Freund K. Mizzer J. Dick W. Thorpe C. Biochemistry. 1985; 24: 5996-6002Crossref PubMed Scopus (55) Google Scholar) were synthesized according to published procedures. The CoA derivatives of 2-octynoic acid, dl-3-hydroxybutyric acid, and 2-hexadecynoic acid were synthesized by the mixed anhydride procedure (11Goldman P. Vagelos P.R. J. Biol. Chem. 1961; 236: 2620-2623Abstract Full Text PDF PubMed Google Scholar). 3-Ketohexadecanoyl-CoA was enzymatically prepared from 2-hexadecynoyl-CoA as described previously (12Thorpe C. Anal. Biochem. 1986; 155: 391-394Crossref PubMed Scopus (52) Google Scholar). Human fetal brain Marathon-Ready cDNA and the Advantage cDNA PCR kit were purchased from CLONTECH. The pGEM-T vector and the Wizard Mini Preps DNA purification system were from Promega.Escherichia coli strain BL21(DE3) pLysS was obtained from Novagen. Restriction endonucleases and T4 ligase were supplied by New England Biolabs Inc. Oligonucleotides were synthesized by Integrated DNA Technologies, Inc. A full-length cDNA of the short chain l-3-hydroxyacyl-CoA dehydrogenase was cloned from human fetal brain Marathon-Ready cDNA according to the manufacturer's procedures. Primer A was synthesized by simulating the active-site sequence of the bovine liver type II enzyme (7Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar), and other primers were designed based on the sequence data obtained in this study. The nucleotide sequences of primers used in the PCR were as follows: primer A, 5′-TTGGAAGCAGAGTATGCAGC-3′; primer B, 5′-CTGCAGGATCCCATATGGCAGCAGCGTGTCGGAGCGTGAAGG-3′; and primer C, 5′-CGGAATTCTATCAAGGCTGCATACGAATGG-3′. The resulting PCR products were cloned into the pGEM-T vector according to the manufacturer's procedures. The DNA nucleotide sequence was determined by the dideoxy method (13Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (57688) Google Scholar), and base determination was automated by using an Applied Biosystems Model 370A DNA sequencer. The cDNA insert (NdeI-EcoRI) containing the whole coding region of human brain short chain l-3-hydroxyacyl-CoA dehydrogenase was removed from the recombinant plasmid pGEM-T-HBHAD by digestion with proper restriction enzymes and subcloned into the NdeI-EcoRI site of the vector pSBETa (a kind gift from Dr. H.-H. Steinbiß) to yield an expression plasmid designated pSBET-HBHAD. The plasmid pSBET-HBHAD was transformed into E. coli BL21(DE3) pLysS by the one-step transformation method (14Chung C.T. Niemela S.L. Miller R.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2172-2175Crossref PubMed Scopus (1199) Google Scholar). The transformants were induced by 0.5 mm IPTG for 6 h. The preparation of cell extract and the purification of the dehydrogenase were performed as described previously (15Yang S.-Y. Li J. He X.-Y. Cosloy S.D. Schulz H. J. Bacteriol. 1988; 170: 2543-2548Crossref PubMed Google Scholar), except for the following modifications. The hydroxylapatite column (2.5 × 4 cm) was equilibrated with 50 mm potassium phosphate (pH 6.6) containing 10% glycerol and 2 mm mercaptoethanol and developed with a gradient made up of 75 ml of this buffer and 75 ml of 0.5 m potassium phosphate (pH 6.6) containing 10% glycerol and 2 mmmercaptoethanol. Fractions (5 ml) were collected and assayed forl-3-hydroxyacyl-CoA dehydrogenase. The recovered enzyme preparation was applied to a smaller hydroxylapatite column (1.5 × 5.5 cm) and eluted with the above-mentioned linear gradient again as a second fractionation step. Protein concentrations were determined by the method of Bradford (16Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223660) Google Scholar). Proteins were separated by SDS-PAGE on a 4–20% gradient gel at pH 8.3 as described previously (17Blackshear P.J. Methods Enzymol. 1984; 104: 237-255Crossref PubMed Scopus (191) Google Scholar). The molecular mass of the purified l-3-hydroxyacyl-CoA dehydrogenase was determined by gel filtration on a Sephadex G-100 column (1 × 50 cm) according to the standard procedure (18Scopes R.K. Protein Purification Principles and Practice. Springer-Verlag New York Inc., New York1994Crossref Google Scholar). The purified enzyme was transferred from SDS-polyacrylamide gel to a polyvinylidene difluoride membrane according to the Bio-Rad protocol, and then the N-terminal sequence was analyzed by automated Edman degradation (19Strickler J.E. Hunkapiller M.W. Wilson K.J. Anal. Biochem. 1984; 140: 533-566Crossref Scopus (31) Google Scholar) using an Applied Biosystems Model 470A gas-phase sequencer. The activity ofl-3-hydroxyacyl-CoA dehydrogenase was determined in the backward direction with acetoacetyl-CoA, 3-ketooctanoyl-CoA, or 3-ketohexadecanoyl-CoA as substrate according to the published procedure (20Binstock J.F. Schulz H. Methods Enzymol. 1981; 71: 403-411Crossref PubMed Scopus (104) Google Scholar). The dehydrogenase assay in the forward direction was performed as described previously (21He X.-Y. Yang S.-Y. Schulz H. Anal. Biochem. 1989; 180: 105-109Crossref PubMed Scopus (36) Google Scholar) using l- andd-3-hydroxybutyryl-CoAs as substrates, respectively. Kinetic parameters of the dehydrogenase were estimated by analysis of the kinetic data with the computer program Leonora (22Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, New York1995Google Scholar). The activities of 17β- and 3α,20β-hydroxysteroid dehydrogenases were measured as described previously (23Jarabak J. Methods Enzymol. 1969; 15: 746-752Crossref Scopus (93) Google Scholar, 24Edwards C.A.F. Orr J.C. Biochemistry. 1978; 17: 4370-4376Crossref PubMed Scopus (26) Google Scholar). The enzyme assay was carried out at 25 °C on a Gilford recording spectrophotometer (Model 2600). One unit of activity is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of substrate to product/min. The nucleotide sequence of the 5′-RACE PCR product (∼0.6 kb) generated from human fetal brain Marathon-Ready cDNA by use of primer A and the adaptor primer 1 was determined by the dideoxy method (13Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (57688) Google Scholar), and then an overlapping 3′-RACE product (∼1 kb) was made by PCR using primer B and the adaptor primer 1. The nucleotide sequence of the 3′-RACE fragment was also determined using the dideoxy method (13Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (57688) Google Scholar). Thereafter, a cDNA fragment (∼0.8 kb) containing the whole coding region of the gene of interest was created by PCR using a pair of primers B and C that were based on sequence data obtained from the 5′- and 3′-RACE products, respectively. A recombinant plasmid (pSBET-HBHAD carrying this cDNA clone was introduced into E. coliBL21(DE3) pLysS cells. Transformants containing plasmids pSBET-HBHAD and pSBETa were induced by IPTG for 6 h. The specific activity ofl-3-hydroxyacyl-CoA dehydrogenase in transformants containing plasmid pSBET-HBHAD was found to be >2 orders of magnitude higher than that in transformants containing a blank vector. Overexpression of the cDNA clone inserted in plasmid pSBET-HBHAD gave rise to a 27-kDa polypeptide band on SDS-PAGE (Fig. 1). The human brain dehydrogenase was purified 6-fold with a recovery of 33% of the original activity by chromatography on hydroxylapatite as summarized in TableI. The purified dehydrogenase was homogeneous as judged by SDS-PAGE (Fig. 1). Since the N-terminal sequence of the purified enzyme determined by Edman degradation perfectly matches the amino acid sequence deduced from the cDNA sequence (Fig. 2) and since the subunit size determined by SDS-PAGE is very close to the calculated molecular mass of 26,923 Da, it is apparent that the cloned cDNA encodes a novel l-3-hydroxyacyl-CoA dehydrogenase from human brain.Table IPurification of human brain short chain l-3-hydroxyacyl-CoA dehydrogenase from E. coli BL21(DE3) pLysS/pSBET-HBHAD induced by IPTGStepTotal activity1-aThe enzyme activities were measured with 27 μm acetoacetyl-CoA as substrate.Total proteinSpecific activityYieldunitsmgunits/mg%Extract56.719.752.87100First hydroxylapatite46.44.0211.5382Second hydroxylapatite18.61.0717.35331-a The enzyme activities were measured with 27 μm acetoacetyl-CoA as substrate. Open table in a new tab Figure 2Sequence of the human short chainl-3-hydroxyacyl-CoA dehydrogenase gene. Exon sequences are in upper-case letters, and introns and flanking sequences are in lower-case letters. The first base of the initiation codon is designated as nucleotide 1. Only the nucleotides of exon sequences are numbered. The lengths of introns in base pairs (bp) are shown in parentheses. The deduced amino acid sequence is indicated by standard one-letter abbreviations under the exon sequence. Amino acid residues are numbered beginning with the initiator methionine as position 1. The N-terminal amino acid sequence determined by Edman degradation is underlined, and it is apparent that the first methionine has been removed presumably by a specific peptidase in E. coli. The termination codon is marked by an asterisk, and the putative polyadenylation signal is double-underlined.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Before the human genomic library was screened with a cDNA fragment of the dehydrogenase, the GenBank Data Bank was searched with the Advance Blast program (25Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61090) Google Scholar) using the human brainl-3-hydroxyacyl-CoA dehydrogenase cDNA sequence as a query sequence. A match with gaps was fortunately found in the human chromosome region Xp11.2. The nucleotide sequence of this 130-kb DNA segment was determined as part of the chromosome X project at the Sanger Center (Hinxton, United Kingdom) and has been submitted by D. Grafham to the GenBank™/EMBL Data Bank (accession no. Z97054). However, it was not known that this piece of DNA contains the humanl-3-hydroxyacyl-CoA dehydrogenase gene. The exon locations were determined by closely scrutinizing the nucleotide sequence in the particular region where the human short chainl-3-hydroxyacyl-CoA dehydrogenase gene was mapped. All exon-intron junctions conform to the GT-AG rule (26Breathnach R. Benoist C. O'Hare K. Gannon F. Chambon P. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4853-4857Crossref PubMed Scopus (902) Google Scholar) (Fig. 2). It was thus found that the direction of transcription of this gene is opposite to that of the KIAA0178 gene and the DNA-binding protein gene, which flank the gene of interest. The human l-3-hydroxyacyl-CoA dehydrogenase gene spans ∼4 kb and consists of six exons and five introns (Fig. 3). The N-terminal amino acid sequence of the gene product was determined by Edman degradation. The results demonstrated that the first ATG codon in frame is the initial codon, and the 5′-untranslated region has a Kozak consensus sequence (27Kozak M. Nucleic Acids Res. 1987; 15: 8125-8131Crossref PubMed Scopus (4549) Google Scholar). Moreover, the sixth exon contains a stop codon (TGA) and a putative polyadenylation signal (Fig. 2). The molecular mass of the purified dehydrogenase was determined by gel filtration on Sephadex G-100 and found to be 108 kDa (Fig. 4). The subunit of this dehydrogenase consists of 261 amino acid residues (Fig. 2), and its subunit size was estimated to be 27 kDa by SDS-PAGE (Fig. 1). Hence, human brainl-3-hydroxyacyl-CoA dehydrogenase is a homotetramer. In contrast, mitochondrial l-3-hydroxyacyl-CoA dehydrogenase from pig heart is composed of two identical 33-kDa subunits (28Birktoft J.J. Holden H.M. Hamlin R. Xuong N.C. Banaszak L.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8262-8266Crossref PubMed Scopus (36) Google Scholar) (Figs.1 and 4). On the basis of certain common structural features, a number of different dehydrogenases constitute the short chain dehydrogenase family (29Persson B. Krook M. Jörnvall H. Eur. J. Biochem. 1991; 200: 537-543Crossref PubMed Scopus (416) Google Scholar). Of 13 largely conserved residues, 6 residues are strictly conserved in all short chain dehydrogenases (29Persson B. Krook M. Jörnvall H. Eur. J. Biochem. 1991; 200: 537-543Crossref PubMed Scopus (416) Google Scholar). Sequence analysis revealed that the human brain dehydrogenase studied here not only has a size similar to other short chain dehydrogenases, but also has all 13 conserved residues (Fig. 5). Therefore, it is concluded that the l-3-hydroxyacyl-CoA dehydrogenase from human brain is a new member of the short chain dehydrogenase family. The primary structure of the human brain dehydrogenase is homologous to bacterial acetoacetyl-CoA reductase (30Peoples O.P. Sinskey A.J. Mol. Microbiol. 1989; 3: 349-357Crossref PubMed Scopus (47) Google Scholar) and 3α,20β-hydroxysteroid dehydrogenase (31Marekov L. Krook M. Jörnvall H. FEBS Lett. 1990; 266: 51-54Crossref PubMed Scopus (68) Google Scholar) with ∼32% identity. The genes of these dehydrogenases are likely to have evolved from a common ancestor gene. Since the homology extends over the full length of the dehydrogenase, it is almost certain that these three dehydrogenases have similar tertiary structures. If so, the subunit of human brain l-3-hydroxyacyl-CoA dehydrogenase would have a single domain with a parallel α/β-structure, as seen in the hydroxysteroid dehydrogenase from Streptomyces exfoliatus 3Formerly Streptomyces hydrogenans. (32Ghosh D. Weeks C.M. Grochulski P. Duax W.L. Erman M. Rimsay R.L. Orr J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10064-10068Crossref PubMed Scopus (234) Google Scholar). Human brain l-3-hydroxyacyl-CoA dehydrogenase is structurally distinct from the well documented mitochondriall-3-hydroxyacyl-CoA dehydrogenases, e.g. pig heart l-3-hydroxyacyl-CoA dehydrogenase, which has a two-domain structure (28Birktoft J.J. Holden H.M. Hamlin R. Xuong N.C. Banaszak L.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8262-8266Crossref PubMed Scopus (36) Google Scholar). The human brain dehydrogenase does not have the GXGXXG fingerprint that exists in the NAD-binding domain of pig heart l-3-hydroxyacyl-CoA dehydrogenase and other medium chain dehydrogenases (4He X.-Y. Yang S.-Y. Biochemistry. 1996; 35: 9625-9630Crossref PubMed Scopus (28) Google Scholar, 28Birktoft J.J. Holden H.M. Hamlin R. Xuong N.C. Banaszak L.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8262-8266Crossref PubMed Scopus (36) Google Scholar, 33Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (1029) Google Scholar), but instead has a GXXXGXG glycine-rich sequence as the marker of the N-terminal NAD-binding region (29Persson B. Krook M. Jörnvall H. Eur. J. Biochem. 1991; 200: 537-543Crossref PubMed Scopus (416) Google Scholar, 32Ghosh D. Weeks C.M. Grochulski P. Duax W.L. Erman M. Rimsay R.L. Orr J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10064-10068Crossref PubMed Scopus (234) Google Scholar). A more remarkable feature of the human brain dehydrogenase is that it does not have the catalytic His-Glu pair identified in otherl-3-hydroxyacyl-CoA dehydrogenases (4He X.-Y. Yang S.-Y. Biochemistry. 1996; 35: 9625-9630Crossref PubMed Scopus (28) Google Scholar, 5He X.-Y. Deng H. Yang S.-Y. Biochemistry. 1997; 36: 261-268Crossref PubMed Scopus (20) Google Scholar). Instead, its C-terminal region contains conserved protic residues, tyrosine 168 and lysine 172, which perhaps play essential roles in the enzyme catalysis similar to tyrosine 152 and lysine 156 of alcohol dehydrogenase from Drosophila melanogaster (34Chen Z. Jiang J.C. Lin Z.-G. Lee W.R. Baker M.E. Chang S.H. Biochemistry. 1993; 32: 3342-3346Crossref PubMed Scopus (142) Google Scholar). The conclusions drawn from the analysis of the human brain dehydrogenase are also applicable to bovine liver type II 3-hydroxyacyl-CoA dehydrogenase (6Kobayashi A. Jian L.L. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar, 7Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar) since there is 89% identity between these two proteins (Fig. 5). Consequently, the bovine liver type II enzyme should also be classified as a short chain dehydrogenase. Based on the structure and organization of the human short chain l-3-hydroxyacyl-CoA dehydrogenase gene, it is concluded that the first three exons of this gene encode the amino-terminal NAD-binding region of the dehydrogenase, whereas the other exons code for the carboxyl-terminal substrate-binding region harboring putative catalytic residues (Figs. 2 and 3). The substrate stereospecificity of this novel enzyme is opposite to that of acetoacetyl-CoA reductase. The human brain dehydrogenase was found to metabolize only l-3-hydroxyacyl-CoA, but not thed-isomer (data not shown). Under the assay conditions used, neither 17β- nor 3α,20β-hydroxysteroid dehydrogenase activity was detected (<0.005 unit/mg). The catalytic properties of the purified human brain dehydrogenase were characterized by steady-state kinetic measurements (35Segel I.H. Enzyme Kinetics, Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme System. Wiley-Interscience, New York1975Google Scholar). To simplify the measurements, the enzyme was usually assayed in the backward direction with 3-ketoacyl-CoA as substrate. The catalytic rate constant for the reduction of acetoacetyl-CoA by NADH at pH 7.0 is 37 ± 1.6 s−1. The apparent K m values for acetoacetyl-CoA and NADH were found to be 89 ± 5.4 and 20 ± 2.8 μm, respectively, and the apparent K m values of the human brain enzyme are 15 ± 1.7 μm for the medium chain substrate and 16 ± 1.0 μm for the long chain substrate (TableII). The kinetic substrate specificity of this enzyme is quite different from those of otherl-3-hydroxyacyl-CoA dehydrogenases. The activity ratio of this dehydrogenase for substrates with C4, C8, and C16 acyl groups was ∼1:2:2 under standard assay conditions, whereas the activity of a mitochondrial matrix l-3-hydroxyacyl-CoA dehydrogenase with short chain substrate was usually greater than that with longer chain substrates. For example, the ratios of l-3-hydroxyacyl-CoA dehydrogenase activities with acetoacetyl-CoA to those with 3-ketooctanoyl-CoA were found to be about 20 and 1.2 for the bovine liver type I and II enzymes, respectively (6Kobayashi A. Jian L.L. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar). It is noteworthy that when acetoacetyl-CoA was used as substrate, the apparent K m values of human brainl-3-hydroxyacyl-CoA dehydrogenase for both the substrate and coenzyme were rather high in comparison with those of bovine liver type II 3-hydroxyacyl-CoA dehydrogenase (6Kobayashi A. Jian L.L. Hashimoto T. J. Biochem. (Tokyo). 1996; 119: 775-782Crossref PubMed Scopus (71) Google Scholar). Moreover, in contrast to pig heart l-3-hydroxyacyl-CoA dehydrogenase being inhibited by acetoacetyl-CoA at concentrations above 25 μm (21He X.-Y. Yang S.-Y. Schulz H. Anal. Biochem. 1989; 180: 105-109Crossref PubMed Scopus (36) Google Scholar), no substrate inhibition was observed with human brain short chain l-3-hydroxyacyl-CoA dehydrogenase (data not shown).Table IIKinetic parameters of human brain short chainl-3-hydroxyacyl-CoA dehydrogenaseSubstratek catK ms−1μmAcetoacetyl-CoA37 ± 1.689 ± 5.4NADH37 ± 1.620 ± 2.83-Ketooctanoyl-CoA28 ± 1.515 ± 1.7NADH28 ± 1.510 ± 1.93-Ketohexadecanoyl-CoA24 ± 1.016 ± 1.0NADH24 ± 1.05.0 ± 1.0 Open table in a new tab During the preparation of this manuscript, a report appeared describing the involvement of a protein called ERAB in neuronal dysfunction associated with Alzheimer's disease (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). ERAB was identified by the yeast two-hybrid system and was shown to bind to a fusion protein containing amyloid β-peptide (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). More ERAB antigen was found in the brains of patients with Alzheimer's disease than in age-matched normal brains by immunoblotting with anti-ERAB peptide antibody (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). The quaternary structure of ERAB is not yet known, and ERAB was said to be a 262-amino acid polypeptide (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar, 36Beyreuther K. Masters C.L. Nature. 1997; 389: 677-678Crossref PubMed Scopus (44) Google Scholar). Only a His-ERAB fusion protein, but not the native protein, has been obtained (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). It is clear that the amino acid sequence of human brain short chain l-3-hydroxyacyl-CoA dehydrogenase is identical to that of ERAB and hence that ERAB is composed of 261 amino acid residues, but not 262 as reported previously. On the basis of the results presented here, it is concluded that ERAB, which previously was suggested to be a steroid dehydrogenase, is actually human brain short chain l-3-hydroxyacyl-CoA dehydrogenase. Hence, the structural and catalytic properties reported here for this novell-3-hydroxyacyl-CoA dehydrogenase should apply to ERAB, which mediates neurotoxicity in Alzheimer's disease (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar, 36Beyreuther K. Masters C.L. Nature. 1997; 389: 677-678Crossref PubMed Scopus (44) Google Scholar). Since the molecular mass of human brain short chainl-3-hydroxyacyl-CoA dehydrogenase determined by SDS-PAGE and gel filtration matches well the value calculated from the amino acid sequence deduced from the cDNA sequence, this enzyme does not seem to undergo post-translational modification. This finding suggests that this novel l-3-hydroxyacyl-CoA dehydrogenase is synthesized in the cytosol, as are many other β-oxidation enzymes. It was reported that ERAB is localized in the endoplasmic reticulum and that the subcellular distribution of ERAB might change in the presence of amyloid β-peptide (8Yan 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. Stern D. Nature. 1997; 389: 689-695Crossref PubMed Scopus (351) Google Scholar). However, since the N-terminal 16 residues of human brain short chain l-3-hydroxyacyl-CoA dehydrogenase are identical to those of the bovine liver mitochondrial type II enzyme (7Furuta S. Kobayashi A. Miyazawa S. Hashimoto T. Biochim. Biophys. Acta. 1997; 1350: 317-324Crossref PubMed Scopus (43) Google Scholar), the human brain dehydrogenase of interest may also have a noncleavable mitochondrial targeting signal sequence, as seen in mitochondrial 3-ketoacyl-CoA thiolase (37Arakawa H. Amaya Y. Mori M. J. Biochem. (Tokyo). 1990; 107: 160-164Crossref PubMed Scopus (15) Google Scholar). It therefore seems likely that this novel l-3-hydroxyacyl-CoA dehydrogenase is transported into mitochondria, as are other β-oxidation enzymes. But it may also be present outside of mitochondria. If so, human brainl-3-hydroxyacyl-CoA dehydrogenase could serve as an intracellular target for amyloid β-peptide (ERAB) and/or function in the elongation of fatty acids at the endoplasmic reticulum. However, we are certain that the newly identified amyloid β-peptide-binding protein is encoded by the human short chainl-3-hydroxyacyl-CoA dehydrogenase gene (Figs. 2 and 3). We have demonstrated in rats that brain mitochondria contain a considerable amount of l-3-hydroxyacyl-CoA dehydrogenase activity (38Yang S.-Y. He X.-Y. Schulz H. J. Biol. Chem. 1987; 262: 13027-13032Abstract Full Text PDF PubMed Google Scholar). We are currently determining the subcellular distribution of human brain short chain l-3-hydroxyacyl-CoA dehydrogenase. We are grateful to Dr. Marshall Elzinga for critical reading of the manuscript.