title: Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch.
abstract: The gene for a membrane-bound, halophilic, and thermostable alpha-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove--the N-C groove--some 30 A away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 A resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with alpha-cyclodextrin and maltoheptaose at 2.2 A resolution, a maltotetraose molecule is bound in subsites -1 to +3, spanning the cleavage point at -1/+1, with the -1 glucosyl residue present as a (2)S(o) skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across -1/+1 and ring distortion. We observe three methionine side chains that serve as "binding platforms" for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.
authors.affiliation: KTH School of Biotechnology, AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
authors.forename: Tien-Chye
authors.initials: TC
authors.lastname: Tan
authors.forename: Benjamin N
authors.initials: BN
authors.lastname: Mijts
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.forename: Bharat K C
authors.initials: BK
authors.lastname: Patel
authors.forename: Christina
authors.initials: C
authors.lastname: Divne
mesh_terms: D020909:Acarbose / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D005936:Glucans / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012965:Sodium Chloride; D013213:Starch / Q000737:chemistry* / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; C031146:maltoheptaose; D012965:Sodium Chloride; D013213:Starch; D000516:alpha-Amylases; D020909:Acarbose