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Publication Abstract from PubMed

Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of the glycoside hydrolase family 66, and it produces cycloisomaltooligosaccharides (CIs). Small CIs (CI-7-9) and large CIs (CI->/=10) are designated as oligosaccharide-type CIs (oligo-CIs) and megalosaccharide-type CIs (megalo-CIs), respectively. CITase from Bacillus circulans T-3040 (BcCITase) produces mainly CI-8 with little megalo-CIs. It has two family 35 carbohydrate-binding modules (BcCBM35-1 and BcCBM35-2). BcCBM35-1 is inserted in a catalytic domain of BcCITase, and BcCBM35-2 is located at the C-terminal region. Our previous studies suggested that BcCBM35-1 has two substrate-binding sites (B-1 and B-2). We implemented site-directed mutagenesis of BcCITase to explore the preference for product size on the basis of the 3D structure of BcCITase. Mutational studies provided evidence that B-1 and B-2 contribute to recruiting substrate and maintaining product size, respectively. A mutant (mutant-R) with four mutations (F268V, D469Y, A513V and Y515S) produced three times as much megalo-CIs (CI-10-12) and 1.5 times as much total CIs (CI-7-12) as compared with the wild-type BcCITase. The 3D structure of the substrate-enzyme complex of mutant-R suggested that the modified product size specificity was attributable to the construction of novel substrate-binding sites in the B-2 site of BcCBM35-1, and reactivity was improved by mutation on subsite -3 on the catalytic domain.

Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity.,Suzuki R, Suzuki N, Fujimoto Z, Momma M, Kimura K, Kitamura S, Kimura A, Funane K Biochem J. 2015 Feb 4. PMID:25649478^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.