Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

Cipolla, Alexandre; D’Amico, Salvino; Barumandzadeh, Roya; Matagne, André; Feller, Georges

doi:10.1074/jbc.m111.274423

Cited by 29 publications

(21 citation statements)

References 40 publications

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“…In this context, cold-active enzymes increase their k cat at the expense of an increase in K m (Feller and Gerday, 2003 ). In fact, stepwise single and multiple mutations engineered on a psychrophilic α-amylase to reconstruct the amino acid substitutions found in a mesophilic homolog exhibit a striking correlation of k cat and K m , such that both decrease concomitanly upon increasing the number of mesophilic residues in the cold-adapted enzyme (Cipolla et al, 2011 ). Nevertheless, some enzymes from psychrophilic organisms that operate under subsaturating substrate concentrations within the cytoplasm exhibit a decrease in this kinetic parameter as an evolutionary strategy for cold adaptation (Bentahir et al, 2000 ; Hoyoux et al, 2001 ; Lonhienne et al, 2001 ).…”

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

“…Psychrophilic and mesophilic alkaline phosphatases are compared to represent changes in the number of insertions and loop extensions, whereas psychrophilic and mesophilic α-amylases are used for visualizing changes in amino acid sequence related to the modification of several properties, listed below each type of amino acid changes. Modified from Helland et al ( 2009 ) and Cipolla et al ( 2011 ).…”

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

“…Typically, the fluorescence quenching constants (as reported by the Stern-Volmer constant) of psychrophilic enzymes are higher than for mesophilic proteins at both low and warm temperatures, thus indicating a more permeable structure (Huston et al, 2008 ; Tang et al, 2012 ), and the variation of fluorescence quenching (i.e., the change in the Stern-Volmer constant) within a temperature range where the native state prevails decreases in the order psychrophilic > mesophilic > thermophilic (D'Amico et al, 2003a ; Georlette et al, 2003 , 2004 ; Cipolla et al, 2012 ), thus indicating that cold-adapted enzymes possess higher flexibility. These experiments can also be combined with mutational analysis to explore the interplay between sequence variation, protein flexibility, and catalytic activity (Cipolla et al, 2011 ; Sigtryggsdóttir et al, 2014 ; Truongvan et al, 2016 ).…”

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

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Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

et al. 2016

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Cold-active enzymes constitute an attractive resource for biotechnological applications. Their high catalytic activity at temperatures below 25 • C makes them excellent biocatalysts that eliminate the need of heating processes hampering the quality, sustainability, and cost-effectiveness of industrial production. Here we provide a review of the isolation and characterization of novel cold-active enzymes from microorganisms inhabiting different environments, including a revision of the latest techniques that have been used for accomplishing these paramount tasks. We address the progress made in the overexpression and purification of cold-adapted enzymes, the evolutionary and molecular basis of their high activity at low temperatures and the experimental and computational techniques used for their identification, along with protein engineering endeavors based on these observations to improve some of the properties of cold-adapted enzymes to better suit specific applications. We finally focus on examples of the evaluation of their potential use as biocatalysts under conditions that reproduce the challenges imposed by the use of solvents and additives in industrial processes and of the successful use of cold-adapted enzymes in biotechnological and industrial applications.

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Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

Section: Evolutionary and Molecular Mechanisms Of The Cold-adaptationmentioning

confidence: 99%

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Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

et al. 2016

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“…On this basis, 17 mutants of this enzyme were constructed, each of them bearing an engineered residue forming a weak interaction found in mesophilic α -amylases but absent in the cold-active α -amylase as well as combinations of up to six stabilizing structural factors [114, 189, 234, 273]. It was shown that these engineered interactions improve all the investigated parameters related to protein stability: the compactness, the kinetically driven stability, the thermodynamic stability, the resistance towards chemical denaturation, and the kinetics of unfolding/refolding.Therefore, these mutants of the psychrophilic α -amylase consistently approximate and reproduce the stability and unfolding patterns of the heat-stable enzymes.…”

Section: Activity-flexibility-stability Relationships: Current Issuesmentioning

confidence: 99%

Psychrophilic Enzymes: From Folding to Function and Biotechnology

2013

Self Cite

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Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

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“…These values have to be compared to 295 kcal mol −1 found for the pig pancreatic α-amylase. In a recent work, two multiple mutants of this cold-adapted α- amylase, derived from the data recorded from single mutations, were also investigated [ 47 ]. The first, Mut5, bears five mutations, previously described, that correspond, as stated above, to structural peculiarities existing in the mesophilic pig pancreatic α-amylase: N150D introduces a salt bridge, V196F restores an aromatic interaction, K300R provides a bidentate interaction with the chloride ion, and T232V reinforces a hydrophobic cluster, as well as Q164I.…”

Section: Engineering Cold-adapted Enzymesmentioning

confidence: 99%

Psychrophily and Catalysis

Gerday

2013

Biology

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Polar and other low temperature environments are characterized by a low content in energy and this factor has a strong incidence on living organisms which populate these rather common habitats. Indeed, low temperatures have a negative effect on ectothermic populations since they can affect their growth, reaction rates of biochemical reactions, membrane permeability, diffusion rates, action potentials, protein folding, nucleic acids dynamics and other temperature-dependent biochemical processes. Since the discovery that these ecosystems, contrary to what was initially expected, sustain a rather high density and broad diversity of living organisms, increasing efforts have been dedicated to the understanding of the molecular mechanisms involved in their successful adaptation to apparently unfavorable physical conditions. The first question that comes to mind is: How do these organisms compensate for the exponential decrease of reaction rate when temperature is lowered? As most of the chemical reactions that occur in living organisms are catalyzed by enzymes, the kinetic and thermodynamic properties of cold-adapted enzymes have been investigated. Presently, many crystallographic structures of these enzymes have been elucidated and allowed for a rather clear view of their adaptation to cold. They are characterized by a high specific activity at low and moderate temperatures and a rather low thermal stability, which induces a high flexibility that prevents the freezing effect of low temperatures on structure dynamics. These enzymes also display a low activation enthalpy that renders them less dependent on temperature fluctuations. This is accompanied by a larger negative value of the activation entropy, thus giving evidence of a more disordered ground state. Appropriate folding kinetics is apparently secured through a large expression of trigger factors and peptidyl–prolyl cis/trans-isomerases.

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Stepwise Adaptations to Low Temperature as Revealed by Multiple Mutants of Psychrophilic α-Amylase from Antarctic Bacterium

Cited by 29 publications

References 40 publications

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

Psychrophilic Enzymes: From Folding to Function and Biotechnology

Psychrophily and Catalysis

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