Jean Louis Arpigny

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.

Cloning and Expression in Escherichia Coli of Three Lipase-Encoding Genes from the Psychrotrophic Antarctic Strain Moraxella Ta144

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.

Molecular adaptations of enzymes from psychrophilic organisms

Abstract The dominating adaptative character of enzymes from cold-evolving organisms is their high turnover number (kcat) and catalytic efficiency ( k cat K m ), which compensate for the reduction of chemical reaction rates inherent to low temperatures. This optimization of the catalytic parameters can originate from the highly flexible structure of these proteins providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Molecular modelling of the 3-D structure of cold-adapted enzymes reveals that only subtle modifications of their conformation can be related to the structural flexibility. The observed structural features include: 1) the reduction of the number of weak interactions involved in the folded state stability like salt bridges, weakly polar interactions between aromatic side chains, hydrogen bonding, arginine content and charge-dipole interactions in α-helices; 2) a lower hydrophobicity of the hydrophobic clusters forming the core of the protein; 3) deletion or substitution of proline residues in loops or turns connecting secondary structures; 4) improved solvent interactions with a hydrophilic surface via additional charged side chains; 5) the occurence of glycine clusters close to functional domains; and 6) a looser coordination of Ca2+ ions. No general rule emerges from the molecular changes observed; rather, each enzyme adopts its own strategy by using one or a combination of these altered interactions. Enzymes from thermophiles reinforce the same type of interactions indicating that there is a continuity in the strategy of protein adaptation to temperature.

Cloning, Sequence and Structural Features of a Lipase from the Antarctic Facultative Psychrophile Psychrobacter Immobilis B10

A lipase gene (lip1) from the facultative psychrophilic strain Psychrobacter immobilis B10 has been cloned and sequenced. The deduced preprotein sequence is composed of 317 amino acids with a predicted M(r) of 35,288. A primary structure alignment of lipases including lip1 shows conserved elements for which a structural role is proposed in the light of recent crystallographic studies. The analysis of the psychrophilic enzyme sequence suggests characteristics in relation with the adaptation to cold.

Molecular adaptations of enzymes from thermophilic and psychrophilic organisms

Although the average temperature on earth is probably of the order of 15 °C, the environments with extreme temperature in which life can be considered possible are very common on the surface of the planet.

Cold enzymes: a hot topic

Chemical reaction rates often show a strong temperature dependency and a decrease of 10°C from room temperature typically divides the rate by a factor oscillating between 1.5 and 4. The decrease of the rate constant k indeed obeys an equation proposed by Svante Arrhenius as early as in 1889:

TEMPERATURE-DEPENDENCE OF GROWTH, ENZYME-SECRETION AND ACTIVITY OF PSYCHROPHILIC ANTARCTIC BACTERIA

K = A{e^{ - Ea/RT}}

Psychrophiles et thermophiles : un problème d’enzyme

(1) in which E a is the activation energy, R the gas constant (8.31 kJ mol−1) and T the temperature in Kelvin.