Enzymes from Cold‐Adapted Microorganisms — The Class C β‐lactamase from the Antarctic Psychrophile Psychrobacter Immobilis A5

Abstract

A heat-labile fl-lactamase has been purified from culture supernatants of Psychrobacter irnmobilis AS grown at 4°C and the corresponding chromosomal ainpC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C P-lactamases. The kinetic parameters of P irnrnobilis p-lactamase for the hydrolysis of some /!-lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three-dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine-mediated H-bonds and aromatic-aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.Keywords: psychrophile ; extremophile ; Antarctic ; P-lactamase ; cephalosporinase.Psychrophilic microorganisms represent a major class of the microbial world if one considers the vast extent of permanently cold environments on Earth (deep-sea waters, polar and alpine regions). In spite of the diversity and abundance of these extremophiles, the numerous physiological and biochemical adaptations to the life at temperatures close to 0°C remain poorly documented [I, 21. The dominating character of cold-adapted enzymes is probably their enhanced turnover number (kcat) and catalytic efficiency (kc.,&C,,,) : improving these kinetic parameters compensates for the reduction of reaction rates at low teniperatures and adequate metabolic fluxes are therefore maintained 131. According to the current hypothesis [4], optimization of the catalytic parameters can originate from the highly flexible structure of these proteins which provides enhanced abilities to undergo conformational changes during catalysis at low temperatures. The usually observed thermal lability is therefore regarded as a consequence of the folded structure flexibility. The molecular origin of this flexibility has been analysed from the primary structure of some psychrophilic bacterial enzymes. All studies suggest a potentially low number of weak interactions stabilizing the folded conformation [ S , 61. We report here the kinetic characterization, the nucleotide sequence and a structural analysis of the P-lactamase secreted by the antarctic psychrophile Psyclzrobacter irnrnobilis A5, a gram-negative bacterial strain collected in an environment ranging in temperature from -20 to +2"C. A brief account of some properties of another P-lactamase from a closely related strain has been given recently 171.

Psychrophilic enzymes: molecular basis of cold adaptation

“…The C-terminal end of A. haloplanctis h-amylase is longer than that of its mesophilic homologues and freely swims in the solvent. The N-terminal h-helix of P. immobilis i-lactamase [48] and the C-terminal helix of salmon trypsin [51] are also less firmly maintained at the surface of the molecule. These examples indicate that loose or relaxed extremities could be involved in the overall weak thermal stability.…”

“…4). To explain this unusual observation, we have argued elsewhere [48] that the mesophilic pathogenes are themselves extremophilic microorganisms (they have evolved under the strong selective pressure of clinical antibiotics) and also that antibiotic concentration in the Antarctic environment is certainly very low. However, another element has to be taken into consideration.…”

“…In addition to the above mentioned crystal structures [50,51,52] models of psychrophilic enzymes such as bacterial subtilisin [59], h-amylase [45], triosephosphate isomerase [60], lipase [61], i-lactamase [48] or fish trypsin [62] and elastase [63] reveal that only subtle modifications of their conformation can be related to the low stability (see also [64,65]). Electrostatic weak interactions and the hydrophobic effect make a lower contribution to the global energy of stabilization of the native state whereas destabilizing forces mainly favour the conformational entropy of the unfolded state.…”

“…Therefore, destabilization can originate from an in-Reviews crease in the number of interactions between water and the molecule in such a way that water molecules compete for the internal H-bonds of the protein [73]. The recurrent observation of very hydrophilic surfaces in psychrophilic enzymes, sometimes leading to abnormal pI, suggests that improved solvent interactions can reduce the compactness of the protein's external shell [48,59,62]. Hydrophobic effect.…”

“…Attempts to evaluate this effect on the basis of structure have been performed, using the hydrophobic index of individual amino acids forming the protein core, or using hydrophobic cluster analysis. Numerous psychrophilic enzymes display substitutions within the hydrophobic core clusters that are accompanied by a sharp decrease of the hydrophobicity index when compared with mesophilic homologues [45,48,51,62]. Entropic factors.…”