Structure and Activity of Malate Dehydrogenase from the Extreme Halophilic Bacteria of the Dead Sea. 1. Conformation and interaction with Water and Salt between 5 M and 1 M NaCl Concentration

Abstract

Large-scale growth of extreme halophilic bacteria from the Dead Sea and purification of malate dehydrogenase (and other proteins) in quantities of hundreds of milligrams makes possible a detailed study of the adaptation to high salt. Halophilic malate dehydrogenase is stable at 20 "C in NaCl solutions between 2.5 -5 M. Below 2.5 M NaCl time-dependent inactivation, paralleled by structural changes, sets in. Within the time scale of the sedimentation, diffusion and circular dichroism experiments discussed here, it was possible to analyze data corresponding to the active halophilic malate dehydrogenase between 1 M and 5 M NaCl. The striking observation was that rather minor conformational changes were observed over the whole range, yet the special properties of the halophilic enzyme seem to be related to its capacity of associating with unusually large amounts of water and of salts, quite distinct from non-halophilic counterparts. These special properties seem to be related to the intact structure of the protein. Some parallel properties of halophilic glutamate dehydrogenase are also discussed.Extreme halophilic bacteria grow best in 20 -30 % NaCl 11 -31. These obligate halophiles are red-pigmented organisms which occur naturally inwaters of high salinity such as theDead Sea, for instance. The extreme halophilic bacteria adapt themIselves to life at high salinity by having internal concentrations (of salt that equal or exceed even the high concentrations in which they grow. They lyse at low salt concentrations. For recent reviews of the unusual properties of the halobacteria, some of which bear closer resemblance to eukaryotes rather than to prokaryotes, we refer to Kushner [4] and to Bailey ,md Morton [5]. Striking compositional properties, which have also been extensively studied in our laboratory are that halophilic proteins possess a molar excess of 16-18 % negatively charged amino acids [6 -81, whereas in non-halophilic proteins the excess is only 7-9%. The content of hydrophobic amino acids is significantly low, yet the content of borderline hydrophobic amino acids (e.g. alanine and threonine) is higher than in non-halophilic counterparts.Halophilic proteins carry out identical functions to their nonhalophilic counterparts, yet in a medium in which one parameter (salt concentration) differs greatly. The halophilic character is an intrinsic property of the proteins and does not depend on additional factors [7,9,10]. The proteins are stabilized by multimolar salt concentrations and the stabilization effect of different salts follows the Hofmeister lyotropic series. At high salt concentrations (3-5 M NaCl or KC1) enzymatic activity of halophilic proteins is observed whereas non-halophilic enzymes tend to be inactivated and to precipitate [11,12]. At low salt concentrations (< 2 M NaCI) halophilic enzymes denature. Denaturation involves unfolding, dissociation and inactivation, processes which may

Haloarcula marismortui (Volcani) sp. nov., nom. rev., an Extremely Halophilic Bacterium from the Dead Sea

“…This strain was originally referred to as "Halobacterium of the Dead Sea." However, the description of the new isolate closely resembled the species description of "Halobacterium marismortui," and Volcani himself agreed that the strain was similar to the original isolate (12). From 1978 on (2) the "Halobacterium of the Dead Sea" was often called "Halobacterium marismortui," although it was never proposed as a neotype strain and was not deposited in a culture collection until recently (as strain ATCC 43049* [T = type strain]) (6).…”

Thermodynamics and the structure of biological macromolecules

“…I would like to emphasize that we do not claim this to be the correct folding process, we only indicate the limitations of the low-resolution methods in providing a satisfactory unique solution in this case. Thus, though this description represents a unified presentation of a From an analysis of sedimentation and diffusion data of hMDH (and also of halophilic glutamate dehydrogenase) we found [133,1341 unusual values for the hydration B , and binding of salt BS ( Table 1). The enzyme hMDH is stable at high concentrations of salt, and becomes unstable upon lowering thc salt concentration below 2.5 M NaCl.…”

Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability

“…Ionic Effects. In comparative experiments to measure salt and water binding to proteins in molar salt solvents, it has been shown that, unlike HmMalDH, BSA does not bind salt but displays usual solvation by about 0.2 g water per g of protein (22). The mild stabilizing effect of molar NaCl solvent on BSA then would be caused by ''salting out'' and would be related to a strengthening of the hydrophobic effect (33).…”

“…It has been shown that, under low-salt conditions, the stabilization of HmMalDH by D 2 O in the solvent arises from a larger entropic contribution to the activation-free energy of unfolding, which indicates a stronger hydrophobic effect than in H 2 O, rather than stronger hydration interactions (16). Because the stabilization of HmMalDH in various solvents has been studied extensively (16,22,23), the protein is well suited for the study of protein dynamics in corresponding conditions to explore a correlation between dynamics and stability. Also, KCl is selected universally as the dominant cytoplasmic salt, and considerable energy is consumed pumping Na ϩ ions out of cells; in addition, it was of particular interest to compare protein dynamics in NaCl and KCl solutions.…”