DOI: 10.3724/SP.J.1206.2013.00031

Progress in Biochemistry and Biophysics (生物化学与生物物理进展) 2013/40:12 PP.1239-1246

Structure and Function Analysis of Calcium Binding Sites in Cyclodextrin Glucanotransferase

Cyclodextrin glucanotransferase (CGTase, EC is an extracellular enzyme capable ofproducing cyclodextrins through an intramolecular transglycosylation reaction. With the application ofcyclodextrins expanding in the industries related to food, pharmaceuticals, cosmetic, etc, CGTase has become thefocus of scientific research nowadays. Calcium binding sites widely exit in α-amylase family. Previous studiesindicated that these sites had very important roles for α-amylase. It was known that CGTases also possess two orthree calcium binding sites. However, their structure and function are not very clear. In the present study, structureand function of calcium binding sites in CGTases were analyzed. Sequence comparisons were performed using theClustalX 1.8 sequence alignment program. Based on the results and crystal structure analysis, it was found thatcalcium binding sites CaⅠ and CaⅡ exist commonly in CGTase. Most amino acids at these two calcium bindingsites are highly conserved, but the residue 29 at CaⅠ and residue 199 at CaⅡ have significant differences betweendifferent types of CGTases. The residue 29 in α-CGTase primarily producing α -cyclodextrin or γ-CGTaseprimarily producing γ-cyclodextrin is Asp, while others are Asn. The residue 199 in γ-CGTase is Ser, while othersare Asp. Calcium binding site Ca Ⅲ only exists in few CGTases. The site consists of residues 315 and 577. Inaddition, site-directed mutagenesis was used to investigate the functions of calcium binding sites in CGTases. Thereplacement of Asp29 by Asn and Arg resulted in 23% and 35% increase in β-cyclization activity, respectively.Mutant D29R and D315A showed higher stability than wild-type CGTase at 60 ℃ . Moreover, the mutant D315Ahad higher β- and γ-cyclodextrin specificity. These results suggested that calcium binding sites might be related tocycling activity, thermal stability, and product specificity of CGTases, which provided the directions for furtherrevealing biological functions of calcium binding sites of CGTases

Key words:cyclodextrin glucanotransferase,calcium binding sites,structure,function

ReleaseDate:2015-04-18 09:14:51

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