CRYSTAL-STRUCTURES OF THE PSYCHROPHILIC ALPHA-AMYLASE FROM ALTEROMONAS-HALOPLANCTIS IN ITS NATIVE FORM AND COMPLEXED WITH AN INHIBITOR

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic s ea-water and it is considered as an extreme psychrophile. We have dete rmined the crystal structures of the alpha-amylase (AHA) secreted by t his bacterium, in its native state to 2.0 Angstrom resolution as well as in complex with Tris to 1.85 Angstrom resolution. The structure of AHA, which is the first experimentally determined three-dimensional st ructure of a psychrophilic enzyme, resembles those of other known alph a-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activat es the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. Th e chloride binding site is situated similar to 5 Angstrom from the act ive site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on t he basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found simil ar to 22 Angstrom from the active site. We found that this triad is eq ually present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in so lution for this cold adapted alpha-amylase.