Cold-adapted enzymes from marine antarctic microorganisms

in Marine Biotechnology (2007), 9(3, May-Jun), 293-304

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the ... [more ▼]

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology. [less ▲]

A nondetergent sulfobetaine prevents protein aggregation in microcalorimetric studies

in Analytical Biochemistry (2006), 352(2), 299-301

Oligosaccharide binding in family 8 glycosidases: Crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product

in Biochemistry (2006), 45(15), 4797-4807

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex ... [more ▼]

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-D-Xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 angstrom resolution) and product xylotriose (at 1.9 angstrom resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apoenzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position Occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking Studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum S-2(O) conformation. [less ▲]

Use of glycoside hydrolase family 8 xylanases in baking

in Journal of Cereal Science (2006), 43(1), 79-84

Xylanases have long been used in the baking industry for improving dough stability and flexibility and for increasing bread volume and crumb structure. Only xylanases from glycoside hydrolase families 10 ... [more ▼]

Xylanases have long been used in the baking industry for improving dough stability and flexibility and for increasing bread volume and crumb structure. Only xylanases from glycoside hydrolase families 10 and I I appear to have been tested in this application and only those from the latter family have as yet found application. Interestingly, enzymes with a putative xylanase activity are also found in glycoside hydrolase families 5, 7, 8 and 43, but apparently these have not, as yet, been tested in baking. Baking trials were used to determine the effectiveness of a psychrophilic and a mesophilic family 8 xylanolytic enzyme as well as a psychrophilic family 10 xylanase and a currently used family 11 commercial mesophilic xylanase. The potential of family 8 xylanases as technological aids in baking was clearly demonstrated as both the psychrophilic enzyme from Pseudoalteromonas haloplanktis TAH3a and the mesophilic enzyme from Bacillus halodurans C-125 had a positive effect on loaf volume. In contrast, the psychrophilic family 10 enzyme from Cryptococcus adeliae TAE85 was found to be ineffective. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase: A microcalorimetric study

in Journal of Biological Chemistry (2005), 280(50), 41307-41314

The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability ... [more ▼]

The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure. [less ▲]

Coping with cold: The genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125

in Genome Research (2005), 15(10), 1325-1335

A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the ... [more ▼]

A considerable fraction of life develops in the sea at temperatures lower than 15 degrees C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome Sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased Solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lipid desaturases achieve both protection against oxygen and synthesis of lipids making the membrane fluid. A remarkable strategy for avoidance of reactive oxygen species generation is developed by A haloplanktis, with elimination of the ubiquitous molybdopterin-dependent metabolism. The A haloplanktis proteome reveals a concerted amino acid usage bias specific to psychrophiles, consistently appearing apt to accommodate asparagine, a residue prone to make proteins age. Adding to its originality, A haloplanktis further differs from its marine Counterparts with recruitment of a plasmid origin of replication for its second chromosome. [less ▲]

The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase

in Journal of Bacteriology (2005), 187(17), 6197-6205

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C ... [more ▼]

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified. fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes. [less ▲]

Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering

in Journal of Molecular Biology (2005), 348(5), 1211-1224

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of ... [more ▼]

Pseudoalteromonas haloplanktis is a psychrophilic Gram-negative bacterium isolated in Antarctica, that lives on organic remains of algae. This bacterium converts the cellulose, highly constitutive of algae, into an immediate nutritive form by biodegrading this biopolymer. To understand the mechanisms of cold adaptation of its enzymatic components, we studied the structural properties of an endoglucanase, Cel5G, by complementary methods, X-ray crystallography and small angle X-ray scattering. Using X-ray crystallography, we determined the structure of the catalytic core module of this family 5 endoglucanase, at 1.4 angstrom resolution in its native form and at 1.6 angstrom in the cellobiose-bound form. The catalytic module of Cel5G presents the (beta/alpha)(8)-barrel structure typical of clan GH-A of glycoside hydrolase families. The structural comparison of the catalytic core of Cel5G with the mesophilic catalytic core of Cel5A from Erwinia chrysanthemi revealed modifications at the atomic level leading to higher flexibility and thermolability, which might account for the higher activity of Cel5G at low temperatures. Using small angle X-ray scattering we further explored the structure at the entire enzyme level. We analyzed the dimensions, shape, and conformation of Cel5G full length in solution and especially of the linker between the catalytic module and the cellulose-binding module. The results showed that the linker is unstructured, and unusually long and flexible, a peculiarity that distinguishes it from its mesophilic counterpart. Loops formed at the base by disulfide bridges presumably add constraints to stabilize the most extended conformations. These results suggest that the linker plays a major role in cold adaptation of this psychrophilic enzyme, allowing steric optimization of substrate accessibility. (c) 2005 Elsevier Ltd. All rights reserved. [less ▲]

Effectiveness of biostimulation techniques for cleaning of diesel polluted subAntarctic soils (Crozet Archipelago)

in Alleman, B. C.; Keley, M. E. (Eds.) In situ bioremediation and on site bioremediation (2005)

Extreme catalysts from low-temperature environments

in Journal of Bioscience & Bioengineering (2004), 98(5), 317-330

Cold-loving or psychrophilic organisms are widely distributed in nature as a large part of the earth's surface is at temperatures around 0 degrees C. To maintain metabolic rates and to prosper in cold ... [more ▼]

Cold-loving or psychrophilic organisms are widely distributed in nature as a large part of the earth's surface is at temperatures around 0 degrees C. To maintain metabolic rates and to prosper in cold environments, these extremophilic organisms have developed a vast array of adaptations. One main adaptive strategy developed in order to cope with the reduction of chemical reaction rates induced by low temperatures is the synthesis of cold-adapted or psychrophilic enzymes. These enzymes are characterized by a high catalytic activity at low temperatures associated with a low thermal stability. A study of protein adaptation strategies suggests that the high activity of psychrophilic enzymes could be achieved by the destabilization of the active site, allowing the catalytic center to be more flexible at low temperatures, whereas other protein regions may be destabilized or as rigid as their mesophilic counterparts. Due to these particular properties, psychrophilic enzymes offer a high potential not only for fundamental research but also for biotechnological applications. [less ▲]