Structural Basis for Thermophilic Protein Stability: Structures of Thermophilic and Mesophilic Malate Dehydrogenases Bjørn Dalhus 1 , Markuu Saarinen 2 , Uwe H. Sauer 2 , Pa ¨r Eklund 2 Kenth Johansson 2 , Andreas Karlsson 2 , S. Ramaswamy 2 Alexandra Bjørk 3 , Bjørnar Synstad 3 , Kristine Naterstad 3 Reidun Sireva ˚g 3 and Hans Eklund 2 * 1 Department of Chemistry University of Oslo Box 1033, Blindern N-0316 Oslo, Norway 2 Department of Molecular Biology, Swedish University of Agricultural Sciences P.O. Box 590, Biomedical Center S-751 24 Uppsala Sweden 3 Department of Biology University of Oslo Box 1066, Blindern N-0316 Oslo, Norway The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been deter- mined by X-ray crystallography and the corresponding structures com- pared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 8C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer–dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer –monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. q 2002 Elsevier Science Ltd. All rights reserved Keywords: thermostability; crystal structure; tetrameric malate dehydrogenases; NAD *Corresponding author Introduction The green gliding bacteria and the green sulfur bacteria represent two distinct groups of photo- tropic bacteria, which are found relatively far apart in the current 16 S rRNA phylogenetic tree. 1 However, the two groups share important charac- teristics, such as the types of chlorophyll and the structure of the antenna systems (chlorosomes). In order to obtain information about structure and thermostability, we have performed comparative studies on malate dehydrogenase (MDH; malate- NAD þ oxidoreductase; EC 1.1.1.37) and its corre- sponding gene, mdh, from several green bacteria: 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved Present addresses: U. H. Sauer, Umea ˚ Center for Molecular Pathogenesis, UCMP, Umea ˚ University, S-90187 Umea ˚, Sweden; S. Ramaswamy, Department of Biochemistry, Bowen Science Building, University of Iowa, Iowa City, IA 52242-1109, USA; B. Synstad, Department of Chemistry and Biochemistry, Agricultural University of Norway, Box 5040, N-1432 A ˚ s, Norway; K. Naterstad, Matforsk, Osloveien 1, N-1430 A ˚ s, Norway. E-mail address of the corresponding author: hasse@xray.bmc.uu.se Abbreviations used: MDH, malate dehydrogenase; LDH, lactate dehydrogenase. doi: 10.1016/S0022-2836(02)00050-5 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 318, 707–721