Journal of Molecular Biology
Regular articleRecognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/Amyloid-β binding alcohol dehydrogenase (ABAD)1
Introduction
Short-chain l-3-hydroxyacyl-CoA dehydrogenases (HADH) catalyse the third step in the fatty acid β-oxidation pathway in mitochondria, converting l-3-hydroxyacyl-Coenzyme A to 3-ketoacyl-coenzyme A with the concurrent reduction of NAD+:
Ketone bodies that are generated by this metabolic process are an important fuel for various organs, particularly the brain when blood glucose levels are low (Torroja et al., 1998). There are three classes of enzyme that catalyse this reaction: (i) the classical type I short chain HADH (HADH I; Noyes & Bradshaw, 1973); (ii) the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein (Uchida et al., 1992); and (iii) type II short chain HADH (HADH II; Furuta et al., 1997). Both type I and II HADH belong to the short-chain dehydrogenase (SDR) family of oxidoreductases and, as they both catalyse the same reaction, might be expected to be very similar. However, type I and type II HADHs differ in molecular mass, subunit association, structure and carbon chain length specificity Kobayashi et al 1996, Furuta et al 1997. Structures of enzymes from the classical HADH I group have been reported Barycki et al 1999a, Barycki et al 1999b. HADH I (also called SCHAD) are usually dimeric enzymes, with a typical subunit molecular mass of 34 kDa, display bilobal subunit structures and have optimal activity against six-carbon substrates. Enzymes from the type II group, on the other hand, differ substantially from the type I enzymes at the amino acid level, where they share no significant sequence homology, and hence are believed to have evolved separately with convergence to catalyse the same reaction. These enzymes are tetrameric, with subunits typically of less than 30 kDa, and display broader substrate specificity. Amino acid sequence homology shows these enzymes are most closely related to hydroxysteroid short-chain dehydrogenases with which they share about 30 % amino acid sequence identity (Figure 1). Both types of enzyme exhibit a similar arrangement of residues in their active sites, including a universally conserved catalytic Tyr/Lys/Ser triad.
This study describes the first structure of a mammalian brain HADH II, termed HADH II/ABAD. The unique features of HADH II/ABAD, as they have emerged from recent work (see references cited below), highlight its broad substrate specificity and the capacity of the enzyme to bind amyloid-β peptide or Aβ. In view of this range of properties, the enzyme has been variously referred to as endoplasmic reticulum amyloid-β binding protein (ERAB; Yan et al., 1997), l-3-hydroxyacyl-CoA dehydrogenase type II (HADH II; He et al., 1998), ABAD (amyloid-β binding alcohol dehydrogenase; Yan et al., 2000a) and SCHAD (He et al., 1999). As the abbreviation SCHAD has also been frequently used for type I short-chain l-3-hydroxyacyl coenzyme A dehydrogenases and ERAB emphasizes the enzyme’s endoplasmic reticulum localization (whereas it is also found in mitochondria; He et al., 1999), we believe that HADH II/ABAD most appropriately describes the enzyme; its potent activity as a type II HADH, its properties as a generalized alcohol dehydrogenase, and its capacity to bind Aβ. HADH II/ABAD forms a homotetramer in solution with a molecular mass of 108 kDa, with each 27 kDa subunit comprising 261 amino acid residues (He et al., 1998). Although the primary activity of HADH II/ABAD appears to be toward l-3-hydroxyacyl-CoA substrates, the marked amino acid homology of these enzymes with hydroxysteroid dehydrogenases is also reflected in their activity. Human HADH II/ABAD (hHADH II/ABAD) has been found to have broad specificity as an alcohol dehydrogenase, oxidising a range of simple alcohols and hydroxy steroids including 17β-estradiol, the precursor of estrogen Yan et al 1999, He et al 1999:
Furthermore, hHADH II/ABAD has been shown to utilize the ketone body β-hydroxybutyrate as a substrate, thereby enhancing the cellular response to metabolic stress (Yan et al., 2000a). In this context, the brains of transgenic mice with targeted neuronal overexpression of hHADH II/ABAD display higher levels of ATP and resistance to ischemic challenge, compared with non-transgenic littermates (Yan et al., 2000a).
Another facet of the biology of HADH II/ABAD that has attracted considerable interest concerns its ability to bind Aβ, the latter believed to contribute to neuronal deterioration underlying Alzheimer’s disease (reviewed by Small & McLean, 1999). Human HADH II has been shown to bind Aβ(1–40) and Aβ(1–42) with a KD of approximately 40–70 nM Yan et al 1997, Yan et al 1999. In an Aβ-rich environment, the properties of HADH II/ABAD appear to undergo a fundamental change; based on studies in cell culture, the enzyme propagates amyloid-induced cellular dysfunction, as suggested by generation of reactive aldehydes and DNA fragmentation (Yan et al., 1999). These initial in vitro observations have recently been extended in vivo using double transgenic mice with targeted overexpression of hHADH II/ABAD and a mutant form of amyloid precursor protein (the latter providing an Aβ-rich environment in the brain) Trillat et al 2000, Yan et al 2000b.
The key to understanding physiologic and pathophysiologic properties of HADH II/ABAD logically begins with structural considerations. In view of the utility of rodent systems as the most practical models for future studies, we have expressed rat [r] HADH II/ABAD, undertaken its kinetic characterization and solved the crystal structure in complex with the NADH/NAD cofactor and two very different substrates: 3-keto-butyrate (acetoacetate), and 17β-estradiol. Rat and human forms of the enzyme share 88 % sequence identity. This is the first crystal structure reported for a type II hydroxyacyl CoA dehydrogenase, and confirms the close relationship between these enzymes and hydroxysteroid short-chain dehydrogenases. A unique feature of HADH II enzymes is the insertion of two additional loop regions between residues 100–110 and 140–150. From the crystal structures it is apparent that the role of these insertions is to support binding of CoA-linked substrates. Despite the very different nature of 3-ketobutyrate and 17β-estradiol, both of these substrates interact with the active-site residues in a very similar manner and are loosely accommodated in an enlarged active site pocket. In view of the 88 % sequence identity between the rat and human forms of HADH II/ABAD, as well as similar enzymatic and Aβ binding properties, we believe that our structural studies of the rat counterpart provide molecular insights highly relevant to the human protein.
Section snippets
Overall structure
Enzymes of the SDR family display a consistent fold and assembly pattern (reviewed by Jornvall et al., 1995) both of which are preserved in the structure of rHADH II/ABAD. SDR enzymes form either dimeric or tetrameric structures; rHADH II/ABAD is a tetramer. Each of the four subunits consists of a single domain formed from a typical, dehydrogenase “Rossmann” fold. Each domain comprises a central β-sheet of seven parallel strands (labelled βA-βG), flanked by six α-helices (labelled αA-αF), three
Expression, purification and crystallisation of rHADH II/ABAD
Rat rHADH II/ABAD (accession number AF049878) was subcloned into pET15b (Novagen) for expression as a His-tagged fusion protein in Escherichia coli BL21 cells. Expression was induced by the addition of 1 mM isopropylthiogalactoside (Melford) for four hours, and the expressed fusion protein was purified using a Ni2+ chelation chromatography column (Pharmacia). rHADH II/ABAD was eluted with a gradient of 0–1 M imidazole, in 500 mM NaCl, 2 mM β-mercaptoethanol, 0.02 % (w/v) azide and 10 % (v/v)
Acknowledgements
We thank the staff at both the Daresbury SRS and EMBL Hamburg Outstation for access to synchrotron data collection facilities, Richard Sessions for assistance with the molecular modelling, and Kay Wilkinson for assistance with crystallization. A.J.P. is supported by a BBSRC studentship. This work is supported by a grant from the BBSRC.
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