Structural Similarities and Evolutionary Relationships in Chloride-Dependent Alpha-Amylases

in Gene (2000), 253(1), 95-105

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha ... [more ▼]

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases. [less ▲]

Cold-Adapted Enzymes: From Fundamentals to Biotechnology

in Trends in Biotechnology (2000), 18(3), 103-7

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography ... [more ▼]

Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes. [less ▲]

Thermodynamic Stability of a Cold-Active Alpha-Amylase from the Antarctic Bacterium Alteromonas Haloplanctis

in Biochemistry (1999), 38(14), 4613-9

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and ... [more ▼]

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state. [less ▲]

Characterization of the C-Terminal Propeptide Involved in Bacterial Wall Spanning of Alpha-Amylase from the Psychrophile Alteromonas Haloplanctis

in Journal of Biological Chemistry (1998), 273(20), 12109-15

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate ... [more ▼]

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein. [less ▲]

Crystal structures of the psychrophilic a-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

in Protein Science : A Publication of the Protein Society (1998), 7(6), 564-572

Structures of the psychrophilic Alteromonas haloplanctis a-amylase give insights into cold adaptation at a molecular level

in Structure (1998), 6

Background: Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degrees C, where the activity of their mesophilic and thermophilic counterparts is drastically ... [more ▼]

Background: Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degrees C, where the activity of their mesophilic and thermophilic counterparts is drastically reduced. It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible. Results: Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic protein's molecular structure. To this' end, we have determined the structure of the recombinant form of a psychrophilic a-amylase from Alteromonas haloplanctis at 2.4 Angstrom resolution. We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 Angstrom resolution, and with available structures of their mesophilic counterparts. These comparative studies have enabled us to identify possible determinants of cold adaptation. Conclusions: We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments. [References: 57] 57 [less ▲]

Psychrophilic Enzymes: Molecular Basis of Cold Adaptation

in Cellular and Molecular Life Sciences : CMLS (1997), 53(10), 830-41

Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the ... [more ▼]

Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes. Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency. This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis. Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues. However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility. [less ▲]

Psychrophiles et thermophiles : un problème d’enzyme

in Chimie Nouvelle (1997), 15

Molecular adaptations of enzymes from psychrophilic organisms

in Comparative Biochemistry and Physiology. A : Comparative Physiology (1997), 118(3), 495-499

The dominating adaptative character of enzymes from cold-evolving organisms is their high turnover number (k(cat)) and catalytic efficiency (k(cat)/K-m), which compensate for the reduction of chemical ... [more ▼]

The dominating adaptative character of enzymes from cold-evolving organisms is their high turnover number (k(cat)) 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 alpha-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 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. (C) 1997 Elsevier Science Inc. [less ▲]

Crystallization and preliminary X-ray diffraction studies of a-amylase from the antarctic psychrophile Alteromonas haloplanctis A23

in Protein Science : A Publication of the Protein Society (1996), 5(10), 2128-2129

A cold-active alpha-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 degrees C. In order to contribute to the understanding of the ... [more ▼]

A cold-active alpha-amylase was purified from culture supernatants of the antarctic psychrophile Alteromonas haloplanctis A23 grown at 4 degrees C. In order to contribute to the understanding of the molecular basis of cold adaptations, crystallographic studies of this cold-adapted enzyme have been initiated because a three-dimensional structure of a mesophilic counterpart, pig pancreatic alpha-amylase, already exists. alpha-Amylase from A. haloplanctis, which shares 53% sequence identity with pig pancreatic alpha-amylase, has been crystallized and data to 1.85 A have been collected. The space group is found to be C222(1) with a = 71.40 A, b = 138.88 A, and c = 115.66 A. Until now, a three-dimensional structure of a psychrophilic enzyme is lacking. [less ▲]