Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus
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© Springer-Verlag 1997
O R I G I N A L PA P E R
Anne Siri Langelandsvik · Ida Helene Steen · Nils-Kåre Birkeland · Torleiv Lien
Properties and primary structure of a thermostable L-malate dehydrogenase from Archaeoglobus fulgidus
Received: 20 November 1997 / Accepted: 28 February 1997
Abstract A thermostable L-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus L-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus L-malate dehydrogenase is the most thermostable L-malate dehydrogenase characterized to date. Addition of K2HPO4 (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to L-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to L-malate dehydrogenase from the archaeon Haloarcula marismortui and other L-lactate-dehydrogenase-like L-malate dehydrogenases. Key words Archaea · Archaeoglobus fulgidus · Thermophiles · Malate dehydrogenase · Lactate dehydrogenase · Thermostable protein · mdh · Glycine motif · Lactate-dehydrogenase-like malate dehydrogenase Abbreviations Tm Melting temperature · EA Activation energy
Introduction Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon (Stetter 1988) oxidizes its substrate, lacA. S. Langelandsvik · I. H. Steen1 · N.-K. Birkeland (Y) · T. Lien Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway Tel. +47-55-582657; Fax +47-55-589671 e-mail: [email protected] Present address: of Medicine, University of Bergen, Harald Hårfagresgt. 1, N-5020 Bergen, Norway Tel. +47-55-582851 e-mail: [email protected]
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tate, via a modified acetyl-CoA pathway and lacks a complete citric acid cycle. However, the cells contain L-malate dehydrogenase activity (Möller-Zinkhan et al. 1989). This enzyme catalyzes the pyridine-nucleotide-dependent interconversion of malate to oxaloacetic acid and is assumed to have a biosynthetic function in A. fulgidus (Danson 1993). Together with L-lactate dehydrogenase, L-malate dehydrogenase is a member of the structurally and functionally homologous family of NAD-dependent 2-ketoacid dehydrogenases (Birktoft et al. 1982). Malate dehydrogenases from a large number of prokaryotes and eukaryotes have been extensively studied. These dehydrogenases are homomeric enzymes organized usually as either dimers or tetramers with subunit molecular masses of 30–38 kDa (Sprott et al. 1979; Sundaram et al. 1980; Grossebüter et al. 1986; Honka et al. 1990; Charnock et al. 1992). Each subunit contains two structurally and functionally different domains, the dinucleotide binding domain and the active-site-containing domain. The former domain is similar in sequence and structure to that of other nicotinamide dinucleotide-dependen
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