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Structural base for enzymatic cyclodextrin hydrolysis.

Stefan Buedenbender1, Georg E Schulz

  • 1Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, D-79104 Freiburg im Breisgau, Germany.

Journal of Molecular Biology
|November 19, 2008
PubMed
Summary
This summary is machine-generated.

Cyclomaltodextrinase enzymes open cyclodextrin rings. Structural studies reveal how Flavobacterium sp. no. 92 enzyme uses Arg464 and Phe274 to bind and hydrolyze cyclodextrins via an induced fit mechanism.

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Area of Science:

  • Enzymology
  • Structural Biology
  • Biochemistry

Background:

  • Cyclodextrins are cyclic oligosaccharides resistant to hydrolysis.
  • Cyclomaltodextrinases are specialized enzymes capable of opening cyclodextrin rings.
  • Understanding the mechanism of cyclodextrin hydrolysis is crucial for various biotechnological applications.

Purpose of the Study:

  • To elucidate the structural basis of cyclodextrin hydrolysis by a cyclomaltodextrinase from Flavobacterium sp. no. 92.
  • To investigate the role of specific amino acid residues, such as Arg464 and Phe274, in substrate binding and catalysis.
  • To characterize the enzyme's mechanism through structural and activity-based studies.

Main Methods:

  • Site-directed mutagenesis of the cyclomaltodextrinase.
  • Crystallization of enzyme-cyclodextrin complexes.
  • X-ray crystallography to determine complex structures.
  • Enzyme activity assays for hydrolysis and transglucosylation.

Main Results:

  • Four distinct complex structures were determined, showing cyclodextrins bound at the active center or outer rim.
  • Arg464 acts as a chaperone, guiding cyclodextrins into the active site.
  • Phe274 induces a conformational change (induced fit) in the bound cyclodextrin, facilitating hydrolysis by Glu340.
  • Activity measurements confirmed the initial ring opening and transglucosylation activities, and effects of mutations.

Conclusions:

  • The study reveals a 'spring-lock' mechanism for cyclodextrin hydrolysis involving substrate-induced conformational changes.
  • Specific residues Arg464 and Phe274 play critical roles in substrate recognition, binding, and positioning for catalysis.
  • Structural insights provide a foundation for enzyme engineering and understanding cyclodextrin metabolism.