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Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic ?-amylase from Antarctic Bacterium.

ABSTRACT: The mutants Mut5 and Mut5CC from a psychrophilic ?-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic ?-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: compactness; kinetically driven stability; thermodynamic stability; resistance toward chemical denaturation, and the kinetics of unfolding/refolding. Concomitantly to this improved stability, both mutants have lost the kinetic optimization to low temperature activity displayed by the parent psychrophilic enzyme. These results provide strong experimental support to the hypothesis assuming that the disappearance of stabilizing interactions in psychrophilic enzymes increases the amplitude of concerted motions required by catalysis and the dynamics of active site residues at low temperature, leading to a higher activity.

SUBMITTER: Cipolla A

PROVIDER: S-EPMC3207396 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic α-amylase from Antarctic Bacterium.

Cipolla Alexandre A D'Amico Salvino S Barumandzadeh Roya R Matagne André A Feller Georges G

The Journal of biological chemistry 20110907 44

The mutants Mut5 and Mut5CC from a psychrophilic α-amylase bear representative stabilizing interactions found in the heat-stable porcine pancreatic α-amylase but lacking in the cold-active enzyme from an Antarctic bacterium. From an evolutionary perspective, these mutants can be regarded as structural intermediates between the psychrophilic and the mesophilic enzymes. We found that these engineered interactions improve all the investigated parameters related to protein stability as follows: comp ...[more]

PMID: 21900238

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Project description:Temperature is one of the most important factors for bacterial growth and development. Cold environments are widely distributed on earth, and psychrotolerant and psychrophilic microorganisms have developed different adaptation strategies to cope with the stress derived from low temperatures. Pseudomonas extremaustralis is an Antarctic bacterium able to grow under low temperatures and to produce high amounts of polyhydroxyalkanoates (PHAs). In this work, we analyzed the genome-wide transcriptome by RNA deep-sequencing technology of early exponential cultures of P. extremaustralis growing in LB (Luria Broth) supplemented with sodium octanoate to favor PHA accumulation at 8°C and 30°C. We found that genes involved in primary metabolism, including tricarboxylic acid cycle (TCA) related genes, as well as cytochromes and amino acid metabolism coding genes, were repressed at low temperature. Among up-regulated genes, those coding for transcriptional regulatory and signal transduction proteins were over-represented at cold conditions. Remarkably, we found that genes involved in ethanol oxidation, exaA, exaB and exaC, encoding a pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the cytochrome c550 and an aldehyde dehydrogenase respectively, were up-regulated. Along with RNA-seq experiments, analysis of mutant strains for pqqB (PQQ biosynthesis protein B) and exaA were carried out. We found that the exaA and pqqB genes are essential for growth under low temperature in LB supplemented with sodium octanoate. Additionally, p-rosaniline assay measurements showed the presence of alcohol dehydrogenase activity at both 8°C and 30°C, while the activity was abolished in a pqqB mutant strain. These results together with the detection of ethanol by gas chromatography in P. extremaustralis cultures grown at 8°C support the conclusion that this pathway is important under cold conditions. The obtained results have led to the identification of novel components involved in cold adaptation mechanisms in this bacterium, suggesting for the first time a role of the ethanol oxidation pathway for bacterial growth at low temperatures.

Dataset Information

Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic ?-amylase from Antarctic Bacterium.

Publications

Stepwise adaptations to low temperature as revealed by multiple mutants of psychrophilic α-amylase from Antarctic Bacterium.

Similar Datasets

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