J. Mol. Bid. (1988) 200, 609-610 Crystallization of Halophilic Malate Dehydrogenase from Halobacterium marismortui ,Malate dehydrogenase from the extreme halophile Halobacterium marismor’tui crystallizes in highly concentrated phosphate solution in space group I2 with cell dimensions a= 113.8 A, b = 122.8 8, c = 126.7 A, fi = 98.1”. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell. Halobacterium marismortui lives in the Dead Sea, the saltiest body of water on earth. The intra- cellular components of this organism are exposed to near-saturated salt concentrations. Proteins from halophilic sources have evolved a special adaptation mechanism that enables them to function at high salinity. Work in this area has recently been reviewed (Werber et al., 1986; Eisenberg & Wachtel, 1986). Knowledge of the three-dimensional structure of malate dehydrogenase from Halo- bacterium murismortui (hMDH)t and its comparison to structures of non-halophilic malate dehydro- genases might shed light on haloadaptation. Structural studies of several other halophilic macromolecules from the same organism have been carried out by us: the ribosomal 50 S particle (Makowski et aZ., 1987); several ribosomal proteins (Shoham et al., 1986); and 2Fe-2S ferredoxin (Sussman et al., 1986). hMDH can be kept stable and active for several years in 4 to 4.3 M-NaCl at neutral pH at room temperature. Tt is, however, degraded in low salt, a process that is accompanied by the dissociation of the dimeric enzyme into subunits as well as the loss of all cc-helical conformation (Mevarech et al., 1977; Mevarech 8r. Neumann, 1977; Pundak & Eisenberg, 1981). As in the case of other halophilic proteins, hMDH has a marked excess of acidic over basic amino acid residues. 19.9 mol 0% (Mevarech et al., 1977), as compared t*o e.g. beef heart cytoplasmic malate dehydrogenase (6 mol %: Siegel & Englard, 1962). Uhough hMDH is an enzyme from an archae- bacterium it, has a higher molar mass (87,000) (Pundak 62 Eisenberg, 1981) than its eukaryotic counterparts, i.e. beef heart mitjochondrial MDH (65,000: Siegel & Englard, 1962), porcine heart c$oplasmic~ MDH (72.000) and porcine heart mitochondrial MDH (68,000: Tsernoglou et al., 1972). .\I1 these enzymes are composed of two chemically identical subunits. The refined crystal struct’ure of porcine heart cytoplasmic MDH showed the two subunits to be structurally nearly identical (Kirktoft & Hanaszak, 1983). t Abbreviation used: h.MDH, malate dehydrogenase from H. nrnrismortui. Analysis ofultracentrifugation and light-scattering data obtained in concentrated NaCl solutions disclosed that hMDH associates with an unusually large amount of water and salt (Pundak & Eisenberg, 1981). This observation was confirmed by a recent combined analysis of neutron and small angle X-ray scattering with ultracentrifugation data (Zaccai et al., 1986a) yielding O-87( kO.07) g water/g protein and O-35( kO.08) g NaCl/g protein. For comparison, the corresponding values for a typical non-halophilic protein, bovine serum albumin, are 0.23 g water/g protein and 0.012 g NaCl/g protein. The radius of gyration of the hMDH particle was found to be about 32 A (1 a = 0.1 nm) (Reich et al., 1982), whereas that calculated for the protein moiety is about 28 A and that of the associated water and salt distribution about 40 il (Zaccai et al., 1986a). Although acidic residues are known to bind about seven molecules of water, whereas other residues bind two to four water molecules (Kuntz, 1971), the excess of negatively charged residues in hMDH cannot solely be responsible for the ability of hMDH to retain such large amounts of water and salt. This ability is lost upon denaturation in low salt (Pundak et al., 1981; Zaccai et al., 19866). In this work, we report the crystallization of hMDH. The enzyme was dialysed against 2 M- phosphate buffer (pH 7-O) and crystallized by vapour diffusion in hanging drops against a reservoir solution of 2.6 M-phosphate at room temperature. Large crystals, up to 1 mm in their longest dimension, developed within several weeks. Preliminary X-ray studies on a Rigaku AFC5-R rotating anode diffractometer indicate the crystals to be monoclinic, space group 12, with a = 113.8( kO.3) A, b = 122.8( kO.3) A, c = 126.7( LO.8) 8, B = 98.1( LO.4)“. (This is equivalent to space group C2 with a = 158.0 w and j?= 134.5”.) The crystals diffract to 2.5 A and survive for about 24 hours in the X-ray beam using a Rigaku rotating anode generator operated at 15 kW when cooled to -0°C. They last about half as long at room temperature. We found (Zaccai et al., unpublished results) the enzyme to be active in phosphate buffer, with a maximum of activity around 0.5 M, decreasing with cX,22-283s/88iO70c~2 $03.00/O 609 0 1988 Academic Press Limited