Differences in the Oligomeric States of the LDH-like L-MalDH from the Hyperthermophilic Archaea Methanococcus jannaschii and Archaeoglobus fulgidus D. Madern,* ,² C. Ebel, ² H. A. Dale, ‡ T. Lien, ‡ I. H. Steen, ‡ N.-K. Birkeland, ‡ and G. Zaccai ² Laboratoire de Biophysique Mole ´ culaire, Institut de Biologie Structurale, UMR 5075, CEA-CNRS-UJF, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France, and Department of Microbiology, UniVersity of Bergen, Jahnebakken 5, P.O. Box 7800, N-5020 Bergen, Norway ReceiVed January 25, 2001; ReVised Manuscript ReceiVed April 24, 2001 ABSTRACT: L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD- dependent enzymes. To gain insight into molecular relationships within this family, we studied two hyperthermophilic (LDH-like) L-MalDH (proteins with LDH-like structure and MalDH enzymatic activity) from the archaea Archaeoglobus fulgidus (Af) and Mehanococcus jannaschii (Mj). The structural parameters of these enzymes determined by neutron scattering and analytical centrifugation showed that the Af (LDH- like) L-MalDH is a dimer whereas the Mj (LDH-like) L-MalDH is a tetramer. The effects of high temperature, cofactor binding, and high phosphate concentration were studied. They did not modify the oligomeric state of either enzyme. The enzymatic activity of the dimeric Af (LDH-like) L-MalDH is controlled by a pH-dependent transition at pH 7 without dissociation of the subunits. The data were analyzed in the light of the crystallographic structure of the LDH-like L-MalDH from Haloarcula marismortui. This showed that a specific loop at the dimer-dimer contact regions in these enzymes controls the tetramer formation. The L-malate dehydrogenases catalyze the NAD(P)- dependent interconversion of oxaloacetate to malate. Analysis of various genome sequences have suggested that this activity is achieved by two nonhomologous groups of enzymes (1). In the first group, proteins purified from Methanothermus ferVidus (Mf), 1 Methanobacterium thermoautotrophicum (Mt), and Methanococcus jannaschii (Mj) present malate dehydrogenase activity (2-4). So far, however, precise information about the oligomeric state, phylogeny, and three- dimensional structure of these proteins is lacking. The attribution of the Mj protein (ORF 1425) as a MalDH remains controversial, since it was demonstrated that it can act on substrates other than malate or oxaloacetate (4). Enzymes from the second group are incomparably better defined. They are members of a large NAD-dependent dehydrogenase family including L-lactate dehydrogenase (L- LDH) and alcohol dehydrogenase (ADH). A number of these L-MalDH have been purified, characterized, and sequenced from a wide variety of organisms (5). Solution studies have shown that they exist as dimeric or tetrameric strucures (6, 7). Various crystallographic structures of bacterial and eucaryal dimeric L-MalDH have been described (refs 8 and 9 and references therein). Their phylogeny has shown that they cluster within the two large mitochondrial and cytosolic clades of L-MalDH (10). For the tetrameric L-MalDH, the crystallographic information is so far limited to a single Archaeal L-MalDH (11, 12). The enzymatic mechanism of L-MalDH has been probed by using site-directed mutagenesis to modulate substrate specificity (13, 14) and coenzyme preference (15). A structure that mimics the catalytic state has been trapped showing that a charge imbalance inside the catalytic vacuole is responsible for substrate discrimina- tion (16). The activity of the monomeric state remains controversial (17-19). However, a dimeric species that condenses to form the tetrameric L-MalDH has been char- acterized to be active (20). Various biochemical studies and primary sequence determinations have suggested that a specific LDH-like group of enzymes within the L-MalDH might be considered (13, 21-25). To refine our understanding of the LDH-like L-MalDH we tried to establish if a strict oligomeric state/primary sequence correlation exists within this group. To achieve this, we used the recombinant hyperthermophilic LDH-like L- MalDH of Methanococcus jannaschii (Mj, ORF 0490) (25) and Archaeoglobus fulgidus (Af)(24). The oligomeric states of the Af and Mj (LDH-like) L-MalDH were determined in solution, at ambient temperature and 70 °C, with the aim to record if these enzymes displayed a thermal-dependent oligomeric state change as it was reported for various hyperthermophilic enzymes (26, 27). This was done in analytical centrifugation (AUC) and small-angle neutron scattering (SANS) experiments, in order to overcome pos- sible temperature or salt artifacts associated with the oligo- meric state determination by size exclusion chromatography. * Corresponding author. Fax: (33).(0).4.38.78.54.94. Tel: (33).(0).4.38.78.95.71. E-mail: madern@ibs.fr. ² Institut de Biologie Structurale. ‡ University of Bergen. 1 Abbreviations: L-MalDH, L-malate dehydrogenase (EC 1.1.1.37); L-LDH, L-lactate dehydrogenase (EC 1.1.1.27); AUC, analytical ultra- centrifugation; CD, circular dichroism; SANS, small-angle neutron scattering; Hm, Haloarcula marismortui; Af, Archaeoglobus fulgidus; Mj, Methanococcus jannaschii; Mf, Methanothermus ferVidus; Tm, Thermotoga maritima; ADH, alcohol dehydrogenase. 10310 Biochemistry 2001, 40, 10310-10316 10.1021/bi010168c CCC: $20.00 © 2001 American Chemical Society Published on Web 08/01/2001