The Multisubstrate Specificity and the Quaternary Structure of Cyclodextrin-/Pullulan-degrading Enzymes.

Abstract

Multiple amino acid sequence alignment and crystal structures of cyclodextrin (CD)-/pullulandegrading enzymes revealed the presence of an N-terminal extension of approximately 130 residuesnot found in ordinary α-amylases. Based on the results obtained from size exclusion chromatogra-phy and sedimentation equilibrium ultracentrifugation experiments, most of these enzymes aremainly in monomer-dimer-tetramer or monomer-dimer-dodecamer equilibrium and the unique Nterminal domain contributes to the formation of oligomeric state in this group of enzymes. Thestudies on the oligomerization and activity of the CD-/pullulan-degrading enzymes indicated thatthere was a significant correlation between the presence of monomer-dimer equilibrium and enzymatic activity. They also suggested that both the monomer and dimer were enzymatically active, but with different substrate preference for fl-CD and starch. The substrate preference of a dimerfor fl-CD was interpreted from the geometry of the active site in the homodimer, that is, small andcompact substrate molecules such as fl-CD might effectively access to the deep and narrow activesite of the dimeric enzyme. Therefore, it was suggested that the monomer-dimer equilibrium present in the reaction mixture reflected different multisubstrate specificity of CD-/pullulan-degrading Multiple amino acid sequence alignment and crystal structures of cyclodextrin (CD)-/pullulan degrading enzymes revealed the presence of an N-terminal extension of approximately 130 residues not found in ordinary α-amylases. Based on the results obtained from size exclusion chromatography and sedimentation equilibrium ultracentrifugation experiments, most of these enzymes are mainly in monomer-dimer-tetramer or monomer-dimer-dodecamer equilibrium and the unique Nterminal domain contributes to the formation of oligomeric state in this group of enzymes. The studies on the oligomerization and activity of the CD-/pullulan-degrading enzymes indicated that there was a significant correlation between the presence of monomer-dimer equilibrium and enzymatic activity. They also suggested that both the monomer and dimer were enzymatically active, but with different substrate preference for β-CD and starch. The substrate preference of a dimer for β-CD was interpreted from the geometry of the active site in the homodimer, that is, small and compact substrate molecules such as fl-CD might effectively access to the deep and narrow activesite of the dimeric enzyme. Therefore, it was suggested that the monomer-dimer equilibrium present in the reaction mixture reflected different multisubstrate specificity of CD-/pullulan-degrading enzymes.