ELSEVIER FEMS Microbiology Letters 134 (1995) 85-90 Isocitrate dehydrogenases from Haloferax volcanii and Sulfolobus solfataricus: enzyme purification, characterisation and N-terminal sequence M6nica L. Camacho a, Richard A. Brown b, Maria-Jo& Bonete ‘, Michael J. Danson b, David W. Hough b.* ’ Dil,i.sibn de Bioquimica, Facultad de Ciencias, Unkersidad de Alicante, Ap. 99, 03080 Alicante , Spain b School zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Biologv and Biochemist?. University qf Bath, Clacerton Down. Bath BA2 7AY, UK Received 3 August 1995; revised 2 October 1995: accepted 2 October 1995 Abstract The isocitrate dehydrogenases from the extremely halophilic Archaeon, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Huloferux dcnnii, and from the hyperther- mophilic Archaeon, Sulfolobus solfaturicus, have been purified to electrophoretic homogeneity. The purified enzymes have been characterised with respect to their cofactor specificities, subunit compositions and their salt and thermal stabilities. N-terminal amino acid sequences have been determined for both enzymes, and multiple alignments with sequences of bacterial and eukaryotic isocitrate dehydrogenases show that the archaeal enzymes most closely resemble the NADP-linked dimeric isocitrate dehydrogenases from the Bacteria. Keywords: Isocitrate dehydrogenase; Archaea; Halophile; Thennophile; Purification; Stability 1. Introduction The citric acid cycle enzyme, isocitrate dehydro- genase (EC 1.1.1.41 and EC 1. I. 1.42), catalyses the oxidative decarboxylation of isocitrate [ 11: Isocitrate + NAD( P) + -+ 2 - oxoglutarate f NAD(P)H + H+ + CO, A number of isoenzymes of isocitrate dehydro- genase, distinguished by their cofactor requirement and subcellular localization, have been identified. In * Corresponding author. Tel.: +44 (1225) 826 867; Fax: + 44 (I 225) 826 449. Eukarya, isocitrate dehydrogenase exists in two forms: an NAD-linked enzyme found only in the mitochondrion and displaying allosteric properties, and a non-allosteric NADP-linked isoenzyme that is found in both the mitochondrion and cytoplasm. Most Bacteria possess only an NADP-isocitrate de- hydrogenase, although NAD-linked and dual-cofac- tor-specific (NAD and NADP) bacteria1 enzymes are known. The gene encoding isocitrate dehydrogenase has been cloned and sequenced from a variety of eu- karyal and bacterial sources. In addition, the crystal structure of the NADP-linked enzyme from Es- cherichia coli has been solved to 2.5 A in both liganded and unliganded forms [2], and references 037% 1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDl 037% 1097(95)00388-6 Downloaded from https://academic.oup.com/femsle/article-abstract/134/1/85/524595 by guest on 02 June 2020