Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41.
The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.
Davail
S
S
Laboratory of Biochemistry, University of Liege, Belgium.
Feller
G
G
Narinx
E
E
Gerday
C
C
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013816:Thermodynamics; D014462:Ultrafiltration
D013381:Subtilisins