Halophilic enzymes: proteins with a grain of salt

Abstract

Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1–4 M range, for activity and stability, and a high excess of acidic over basic amino residues. The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe–2S protein ferredoxin. It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein.

Introduction

The title of the Ph.D. thesis of the late Professor Benjamin Elazari-Volcani, submitted to the Hebrew University in Jerusalem in 1940, is: ‘Studies on the Microflora of the Dead Sea’. This pioneering work completely shattered the generations-old myths of total sterility of the Dead Sea by showing that this unusual lake, located at the lowest point of terrestrial earth, is the breeding ground for numerous prokaryotic and unicellular eukaryotic species. The most abundant organisms in the Dead Sea are the extremely halophilic archaea, generally called halobacteria, whose cell density can reach 10⁷ cells/ml. Halobacteria which are obligatory halophiles, can only grow in media containing salt concentrations of 1.5 M or higher. Besides the Dead Sea, these archae also flourish in natural salt lakes such as the Great Salt Lake in Utah, in artificial sea water pools made for the purpose of salt extraction, in spoiled salted fish and surprisingly enough, embedded in rocks in salt mines [1].

In order to overcome the extreme osmotic pressure of these hypersaline environments, halophilic bacteria and eukaryotes accumulate mostly neutral organic compatible solutes and exclude most of the inorganic salts. In contrast, the halophilic archaea balance the external high salt concentration by intracellular accumulation of inorganic ions to concentrations that exceed that of the medium. Therefore, all the cellular components of the halophilic archaea must be adapted to function at the extremely high intracellular salt concentration.

The effect of salt concentration on biopolymers has been reviewed recently [2], [3]. In general, the final conformation of a biopolymer depends on the intramolecular interactions between surfaces of the polymer, on intermolecular interactions between surfaces of the polymer with surfaces of other polymers, and the interactions of the surface of the biopolymer with water and solutes. These interactions include those that are hydrophobic in nature, as well as hydrogen bonds and electrostatic attraction and repulsion. At low salt concentrations (<0.1 M), only the conformations of highly charged biopolymers are affected. At high salt concentrations (>0.1 M), salts affect the conformation of biopolymers by reducing the effective concentration of water and by interacting specifically with the surface of the biopolymer. Some ions are excluded from the biopolymer surface and their effect is mainly through reducing the activity of water. The nature of the interactions of different ions with proteins is defined by the ‘Hoffneister Series’. Some ions, such as sulfate, phosphate and ammonium, promote the folding of proteins, intermolecular association and even aggregation while some ions, such as iodide and guanidinium, promote dissociation of protein complexes and aggregates and the unfolding of protein chains.

The dependence of the stability and the catalytic properties of halobacterial proteins on salt concentration has been the subject of studies for many years (for reviews see Lanyi [4] and Eisenberg et al. [5]). In the following communication, we shall review the structural and biochemical properties of two halobacterial proteins whose three-dimensional structures have been determined by X-ray crystallography: (1) the enzyme malate dehydrogenase (hMDH) from Haloarcula marismortui; and (2) the 2S–2Fe ferredoxin (HmFd) of the same archaeon. Early studies on these proteins were performed in the laboratory of Professor Eisenberg, whose 17 years of service on the editorial board of Biophysical Chemistry is recognized by the special edition of this issue.