Crystal structure of a maltotetraose-formingexo-amylase from Pseudomonas stutzeri

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• 作者：Morishita ; Yoshihiro ; Hasegawa ; Kazuya ; Matsuura ; Yoshiki ; Katsube ; Yukiteru ; Kubota ; Michio ; et. al.
• 关键词：crystal structure ; Pseudomonas stutzeri ; maltotetraose forming exo-amylase ; calcium-binding ; (β ; /α ; )₈ barrel ; G4-amylase ; 1 ; 4-α ; -d-glucan maltotetraohydrolase ; MIR ; multiple isomorphous replacement ; CSA ; contact surface area ; TAA ; T
• 刊名：Journal of Molecular Biology
• 出版年：1997
• 出版时间：April 4, 1997
• 年：1997
• 卷：267
• 期：3
• 页码：661-672
• 全文大小：552 K

文摘

The three-dimensional structure of an exo-type α-amylase from Pseudomonas stutzeri which degrades starch from its non-reducing end to produce maltotetraose has been determined by X-ray structure analysis. The catalytic domain of this enzyme (G4-2), whose structure was determined, is a product of spontaneous limited proteolysis in culture broth. It has 429 amino acid residues and a molecular mass of 47,200, and crystallizes in ammonium sulfate solution at pH 7.5. The structure was elucidated by the multiple isomorphous replacement method and refined at 2.0 Å resolution, resulting in a final R-factor of 0.178 for significant reflections with a root-mean-square deviation from ideality in bond distances of 0.013 Å. The polypeptide chain of this molecule folds into three domains; the first with a (β/α)₈ barrel structure, the second with an excursed part from the first one, and the third with five-stranded antiparallel β-sheets. The active cleft is formed on the C-terminal side of the β-sheets in the (β/α)₈ barrel as in the known endo-type α-amylases. A histidine side-chain nitrogen ND1 is coordinated to one of the bound calcium ion. The recognition site of the non-reducing end of the amylose that determines exo-wise degradation is presumed to be at one end of this cleft where there is a disordered loop consisting of the 66th to 72nd residues, and a loop carrying an aspartic acid (Asp160). These structural features may be responsible for the binding of the non-reducing end of the substrate amylose.