Protein Science, Vol 7, Issue 3 564-572, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

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

N. AGHAJARI, G. FELLER, C. GERDAY and R. HASER
Laboratoire d'Architecture et Fonction des Macromolecules Biologiques, UPR9039, Institut de Biologie Structurale et Microbiologie, IFR1, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France Present address: Institut de Biologie et Chimie des Proteines, UPR412, CNRS, 7 Passage du Vercors, 69367 Lyon Cedex 07, France.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the {alpha}-amylase (AHA) secreted by this bacterium, in its native state to 2.0 A resolution as well as in complex with Tris to 1.85 A resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known {alpha}-amylases of various origins with a surprisingly greatest similarity to mammalian {alpha}-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate {alpha}-1,4-glycosidic bonds. The chloride binding site is situated ~5 A from the active 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 the 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 ~22 A from the active site. We found that this triad is equally present in other chloride dependent {alpha}-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted {alpha}-amylase.
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