Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Area of Science:

• Enzymology
• Protein Chemistry
• Industrial Biotechnology

Background:

• Cyclodextrin glycosyltransferases (CGTases) are vital industrial enzymes for producing cyclomaltooligosaccharides (cyclodextrins).
• Understanding CGTase structure-function relationships is key to optimizing cyclodextrin production.
• Bacillus circulans CGTase DF 9R is an industrially relevant enzyme with potential for detailed structural analysis.

Purpose of the Study:

• To elucidate the structural features and structure-function relationship of Bacillus circulans DF 9R cyclodextrin glycosyltransferase.
• To identify critical amino acid residues involved in the catalytic activity and substrate binding of CGTase.
• To characterize the role of specific histidine residues and the conserved -6 subsite in enzyme function.

Main Methods:

• Protein ethoxyformylation to identify reactive amino acid residues.
• Radiolabeling with (14)C to track modified residues.
• Tryptic digestion and peptide mixture analysis to pinpoint modified sites.
• Sequence analysis and comparison with known CGTases.

Main Results:

• Ethoxyformylation modified only one histidine residue, drastically reducing enzyme activity.
• Histidine 233 was identified as the most reactive site for ethoxyformylation.
• The enzyme possesses a glutamine at position 179 in the conserved -6 subsite, differing from the previously required glycine for substrate binding.
• The identified CGTase exhibits characteristics of an alpha- or alpha/beta-cyclodextrin producer.

Conclusions:

• Histidine 233 is critical for the cyclizing and hydrolytic activity of Bacillus circulans CGTase.
• The presence of glutamine at position 179 challenges the existing induced-fit model for substrate binding at the -6 subsite.
• This research provides novel insights into CGTase structure-function, aiding in the development of improved cyclodextrin production processes.