Structural Evolution of Enzymes from Antarctic Psychrotrophs and Molecular Mechanism for Their Higher Activities at Low Temperatures

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Enzymes produced by cold-adapted microorganisms generally exhibit higher catalytic efficiency at low temperatures and lower thermostability than their mesophilic counterparts. We here report on the subtilisin-like alkaline serine protease Apa2 secreted by Antarctic psychrotroph Alteromonas sp. AS-11, which is characterized by not only a higher proteolytic activity at low temperatures but also a greater thermostability than the mesophilic subtilisin BPN'. The specific proteolytic activity of Apa2 relative to subtilisin BPN' increased with decreasing temperature, although the optimal temperatures of both enzymes were the same. At 0℃, Apa2 was 9.9-fold more active than subtilisin BPN'. It had 10 times longer half-life time at 57℃ than subtilisin BPN' with higher activation enthalpy and entropy for thermal denaturation. These features of Apa2 support the idea that structural determinants , of conformational flexibility required for high catalytic efficiency can be separated from those of thermal instability. The gene apa2 encodes a 629-amino-acid pre-pro-peptide containing a mature enzyme of 283 residues that has significant homologies with subtilisin-like serine proteases. The structural model of Apa2 suggests that the disulfide bonds, a higher number of salt bridges, and Ca^<2+>binding contribute to its thermostability, while its cold-adaptation may be attributed to the increase in structural flexibility at loop and β-turn or bend regions: