3WNP

D308A, F268V, D469Y, A513V, and Y515S quintuple mutant of Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase complexed with isomaltoundecaose

Summary for 3WNP

• Entry DOI: 10.2210/pdb3wnp/pdb
• Related: 3WNK 3WNL 3WNM 3WNN 3WNO
• Descriptor: Cycloisomaltooligosaccharide glucanotransferase, alpha-D-glucopyranose-(1-6)-alpha-D-glucopyranose-(1-6)-alpha-D-glucopyranose-(1-6)-beta-D-glucopyranose, CALCIUM ION, ... (7 entities in total)
• Functional Keywords: c2 type immunoglobulin fold, (beta/alpha)8-barrel, beta-jelly roll, greek key, glycoside hydrolase, alpha-1, 6-glucan, transferase
• Biological source: Bacillus circulans
• Total number of polymer chains: 2
• Total formula weight: 161174.80

Authors

Suzuki, R.,Suzuki, N.,Fujimoto, Z.,Momma, M.,Kimura, K.,Kitamura, S.,Kimura, A.,Funane, K. (deposition date: 2013-12-10, release date: 2014-02-05, Last modification date: 2023-11-08)

Primary citation

Suzuki, R.,Suzuki, N.,Fujimoto, Z.,Momma, M.,Kimura, K.,Kitamura, S.,Kimura, A.,Funane, K.
Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity.
Biochem.J., 467:259-270, 2015

PubMed Abstract: Cycloisomaltooligosaccharide glucanotransferase (CITase) is a member of 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) [Suzuki et al. (2014) J. Biol. Chem. 289, 12040-12051]. 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 (WT) 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.

PubMed: 25649478
DOI: 10.1042/BJ20140860

Experimental method

X-RAY DIFFRACTION (2.8 Å)