Structural Analysis of a Chimeric Bacterial -Amylase. High-Resolution Analysis of Native and Ligand Complexes

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• 作者：Andrzej M. Brzozowski ; David M. Lawson ; Johan P. Turkenburg ; Henrik Bisgaard-Frantzen ; Allan Svendsen ; Torben V. Borchert ; Zbigniew Dauter ; Keith S. Wilson ; and Gideon J. Davies
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 8, 2000
• 年：2000
• 卷：39
• 期：31
• 页码：9099 - 9107
• 全文大小：527K
• 年卷期：v.39,no.31(August 8, 2000)
• ISSN：1520-4995

文摘

Several chimeric mages/gifchars/alpha.gif" BORDER=0>-amylases genes were constructed by an in vivo recombination techniquefrom the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafterBA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, hasbeen extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 Å. The 3-dimensionalstructure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolutionof 1.7 Å. It consists of 483 amino acids, organized similarly to the known B. lichiniformis mages/gifchars/alpha.gif" BORDER=0>-amylasestructure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two ofthese form part of a linear cluster of three ions, the central ion being attributed to sodium. This clusterlies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to themajor Ca²⁺ binding site of fungal mages/gifchars/alpha.gif" BORDER=0>-amylases. The third calcium ion is found at the interface of the Aand C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis mages/gifchars/alpha.gif" BORDER=0>-amylase structure.It is found on the C domain where it bridges the two mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sheets. Three acid residues (Glu261, Asp328, andAsp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, asingle molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the threeactive-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of itsparents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose bindingsite on the N-terminal face of the (mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">/mages/gifchars/alpha.gif" BORDER=0>)₈ barrel of the molecule, not previously described for any mages/gifchars/alpha.gif" BORDER=0>-amylasestructure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharideinhibitor acarbose, at 1.9 Å, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme.The unambiguous presence of three unsaturated rings in the ²H₃ half-chair/²E envelope conformation,adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-deriveddecasaccharide by a two-step transglycosylation mechanism.