Georges Feller

Psychrophilic enzymes: hot topics in cold adaptation

More than three-quarters of the Earths surface is occupied by cold ecosystems, including the ocean depths, and polar and alpine regions. These permanently cold environments have been successfully colonized by a class of extremophilic microorganisms that are known as psychrophiles (which literally means cold-loving). The ability to thrive at temperatures that are close to, or below, the freezing point of water requires a vast array of adaptations to maintain the metabolic rates and sustained growth compatible with life in these severe environmental conditions.

Cold-adapted enzymes: from fundamentals to biotechnology

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.

Psychrophilic microorganisms: challenges for life

The ability of psychrophiles to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently cold environments. These challenges include: reduced enzyme activity; decreased membrane fluidity; altered transport of nutrients and waste products; decreased rates of transcription, translation and cell division; protein cold‐denaturation; inappropriate protein folding; and intracellular ice formation. Cold‐adapted organisms have successfully evolved features, genotypic and/or phenotypic, to surmount the negative effects of low temperatures and to enable growth in these extreme environments. In this review, we discuss the current knowledge of these adaptations as gained from extensive biochemical and biophysical studies and also from genomics and proteomics.

PSYCHROPHILIC ENZYMES : MOLECULAR BASIS OF COLD ADAPTATION

Abstract Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes. Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency. This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis. Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues. However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility.

Psychrophilic enzymes: a thermodynamic challenge

Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0-30 degrees C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold alpha-amylase, beta-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost.

Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility.

Basic theoretical and practical aspects of activation parameters are briefly reviewed in the context of cold-adaptation. In order to reduce the error impact inherent to the transition state theory on the absolute values of the free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation, it is proposed to compare the variation of these parameters between psychrophilic and mesophilic enzymes, namely Delta(DeltaG(#))(p-m), Delta(DeltaH(#))(p-m) and Delta(DeltaS(#))(p-m). Calculation of these parameters from the available literature shows that the main adaptation of psychrophilic enzymes lies in a significant decrease of DeltaH(#), therefore leading to a higher k(cat), especially at low temperatures. Moreover, in all cases including cold-blooded animals, DeltaS(#) exerts an opposite and negative effect on the gain in k(cat). It is argued that the magnitude of this counter-effect of DeltaS(#) can be reduced by keeping some stable domains, while increasing the flexibility of the structures required to improve catalysis at low temperature, as demonstrated in several cold-active enzymes. This enthalpic-entropic balance provides a new approach explaining the two types of conformational stability detected by recent microcalorimetric experiments on psychrophilic enzymes.

Structures of the psychrophilic Alteromonas haloplanctis α-amylase give insights into cold adaptation at a molecular level

BACKGROUND . Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degreesC, where the activity of their mesophilic and thermophilic counterparts is drastically reduced. It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible. RESULTS . Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic proteins molecular structure. To this end, we have determined the structure of the recombinant form of a psychrophilic alpha-amylase from Alteromonas haloplanctis at 2.4 A resolution. We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 A resolution, and with available structures of their mesophilic counterparts. These comparative studies have enabled us to identify possible determinants of cold adaptation. CONCLUSIONS . We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments.

Molecular adaptations to cold in psychrophilic enzymes

Abstract. Psychrophiles or cold-loving organisms successfully colonize cold environments of the Earths biosphere. To cope with the reduction of chemical reaction rates induced by low temperatures, these organisms synthesize enzymes characterized by a high catalytic activity at low temperatures associated, however, with low thermal stability. Thanks to recent advances provided by X-ray crystallography, protein engineering and biophysical studies, we are beginning to understand the molecular adaptations responsible for these properties which appear to be relatively diverse. The emerging picture suggests that psychrophilic enzymes utilize an improved flexibility of the structures involved in the catalytic cycle, whereas other protein regions if not implicated in catalysis may or may not be subjected to genetic drift.

A novel family 8 xylanase, functional and physicochemical characterization

Xylanases are generally classified into glycosyl hydrolase families 10 and 11 and are found to frequently have an inverse relationship between their pI and molecular mass values. However, we have isolated a psychrophilic xylanase that belongs to family 8 and which has both a high pI and high molecular mass. This novel xylanase, isolated from the Antarctic bacteriumPseudoalteromonas haloplanktis, is not homologous to family 10 or 11 enzymes but has 20–30% identity with family 8 members. NMR analysis shows that this enzyme hydrolyzes with inversion of anomeric configuration, in contrast to other known xylanases which are retaining. No cellulase, chitosanase or lichenase activity was detected. It appears to be functionally similar to family 11 xylanases. It hydrolyzes xylan to principally xylotriose and xylotetraose and is most active on long chain xylo-oligosaccharides. Kinetic studies indicate that it has a large substrate binding cleft, containing at least six xylose-binding subsites. Typical psychrophilic characteristics of a high catalytic activity at low temperatures and low thermal stability are observed. An evolutionary tree of family 8 enzymes revealed the presence of six distinct clusters. Indeed classification in family 8 would suggest an (α/α)6fold, distinct from that of other currently known xylanases.

Protein stability and enzyme activity at extreme biological temperatures.

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.