Stability and Structural Analysis of Alpha-Amylase from the Antarctic Psychrophile Alteromonas Haloplanctis A23

in European Journal of Biochemistry (1994), 222(2), 441-7

The alpha-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic alpha-amylase. The psychrophilic alpha-amylase is however ... [more ▼]

The alpha-amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic alpha-amylase. The psychrophilic alpha-amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4 degrees C and a lower conformational stability estimated as 10 kJ.mol-1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity. When compared with the known three-dimensional structure of porcine pancreatic alpha-amylase, homology modelling of the psychrophilic alpha-amylase reveals several features which may be assumed to be responsible for a more flexible, heat-labile conformation: the lack of several surface salt bridges in the (beta/alpha)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd = 44 nM) and for Cl- (Kd = 1.2 mM at 4 degrees C) can result from single amino acid substitutions in the Ca(2+)-binding and Cl(-)-binding sites and can also affect the compactness of alpha-amylase. [less ▲]

THE BLOOD PROTEINS OF THE ANTARCTIC ICEFISH CHANNICHTHYS-RHINOCERATUS - BIOLOGICAL SIGNIFICANCE AND PURIFICATION OF THE 2 MAIN COMPONENTS

in Comparative Biochemistry and Physiology. B : Comparative Biochemistry (1994), 109(1), 89-97

The lack of hemoglobin and of carbonic anhydrase in the blood of icefish suggest that substantial adaptations of the acid-base balance should occur in order to ensure blood pH homeostasis. The level of ... [more ▼]

The lack of hemoglobin and of carbonic anhydrase in the blood of icefish suggest that substantial adaptations of the acid-base balance should occur in order to ensure blood pH homeostasis. The level of peptidic histidyl and of reactive -SH groups per unit of body mass in icefish plasma are 12-13 times higher that those of Notothenia rossii, a common red-blooded Antarctic species. It is proposed that the high level of imidazole ring in icefish plasma improves the non-bicarbonate buffering capacity and that the reactive sulfhydryls are involved om a redox buffer as in some other hypoxia tolerant species. After plasma fractionation on Ultrogel AcA 34, the two main icefish serum proteins have been purified by DEAE cellulose chromatography (IFI) and by HPLC on anion exchange column (IF2). IFI has been identified as a cysteine-rich para-albumin and IF2 as an histidine-rich immunoglobulin-like protein. [less ▲]

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

in Biochimica et Biophysica Acta (1993), 1171(3), 331-3

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 ... [more ▼]

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. [less ▲]

Sequence of the Subtilisin-Encoding Gene from an Antarctic Psychrotroph Bacillus Ta41

in Gene (1992), 119(1), 143-4

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of ... [more ▼]

The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined. The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids. Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site. [less ▲]

Purification, Characterization, and Nucleotide Sequence of the Thermolabile Alpha-Amylase from the Antarctic Psychrotroph Alteromonas Haloplanctis A23

in Journal of Biological Chemistry (1992), 267(8), 5217-21

The alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to ... [more ▼]

The alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to establish the primary structure of the mature A. haloplanctis alpha-amylase which is composed of 453 amino acids with a predicted Mr of 49,340 and a pI of 5.5. Three Ca2+ ions are bound per molecule and its activity is modulated by chloride ions. Within the four consensus sequences, Asp-174, Glu-200, and Asp-264 are the proposed catalytic residues. The psychrotrophic A. haloplanctis alpha-amylase is characterized by a high amylolytic activity at low temperatures, a reduced apparent optimal temperature, and typical thermodynamic activation parameters A. haloplanctis alpha-amylase has also a low thermal stability as demonstrated by the temperature effect on both activity and secondary structure. It is suggested that structure flexibility and lower sensitivity of secondary structure to temperature variations in the low temperature range are the main structural adaptations of the psychrotrophic enzyme. The unusual stacking of small amino acids around the catalytic residues is proposed as a factor inducing active site flexibility and concomitant high activity of the enzyme at low temperatures. [less ▲]

The Lactate Dehydrogenase of the Icefish Heart: Biochemical Adaptations to Hypoxia Tolerance

in Biochimica et Biophysica Acta (1991), 1079(3), 343-7

Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or ... [more ▼]

Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or identical to those of its skeletal muscle counterpart and other M-type lactate dehydrogenases. A model involving a dual oxidative-anaerobic metabolism of the icefish heart is proposed. [less ▲]

Nucleotide Sequence of the Lipase Gene Lip2 from the Antarctic Psychrotroph Moraxella Ta144 and Site-Specific Mutagenesis of the Conserved Serine and Histidine Residues

in DNA & Cell Biology (1991), 10(5), 381-8

The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a ... [more ▼]

The lip2 gene from the antarctic psychotroph Moraxella TA144 was sequenced. The primary structure of the Lip2 preprotein deduced from the nucleotide sequence is composed of 433 amino acids with a predicted Mr of 47,222. This enzyme contains a Ser-centered consensus sequence and a conserved His-Gly dipeptide found in most lipase amino-terminal domains. These sequences are involved in the lipase active site conformation since substitution of the conserved Ser or His residues by Ala and Gln, respectively, results in the loss of both lipase and esterase activities. Structural factors that would allow proper enzyme flexibility at low temperatures are discussed. It is suggested that only subtle changes in the primary structure of these psychrotrophic enzymes can account for their ability to catalyze lipolysis at temperatures close to 0 degrees C. [less ▲]

Sequence of a Lipase Gene from the Antarctic Psychrotroph Moraxella Ta144

in Nucleic Acids Research (1990), 18(21), 6431

LIPASES FROM PSYCHROTROPHIC ANTARCTIC BACTERIA

in FEMS Microbiology Letters (1990), 66(1-3), 239-243

Bioenergetic peculiarity of heart mitochondria from the hemoglobin-and myoglobin-free antartic icefish

in Biochimica et Biophysica Acta-Bioenergetics (1989), 977