Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Abstract

Background: Catalytic residues and molecular mechanism of GH-66 enzymes were hitherto unknown. Results: Novel dextranase produced isomaltotetraose and cyclo-isomaltosaccharides. Its nucleophile (Asp 340 ) and acid/base catalyst (Glu 412 ) were identified by a chemical rescue approach. Conclusion: Three GH-66 enzyme types were newly classified for the first time. Significance: This work elucidates production of isomaltotetraose and cycloisomaltosaccharides, classification of GH-66, identification of catalytic residues, and novel dextran-forming type chemical rescue.

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

“…Recently, both Bacteroides thetaiotaomicron dextranase lacking this inserted domain and Paenibacillus sp. endo-dextranases containing the CBM35 domain have been reported to show CI producing activity, although the activity of these two enzymes is reduced when compared with the activity of BcCITase (48,49). Furthermore, deletion mutants, in which BcCBM35-1 is removed from BcCITase, still retain approximately one-twentieth of the CI producing activity of the wildtype enzyme (21).…”

“…In GH66, methionine is conserved only in CITases, whereas this residue is replaced by isoleucine in streptococcal dextranases and a variety of hydrophobic residues are found in other enzymes. Paenibacillus dextranase has tryptophan at this position and synthesizes relatively large CI molecules (48). Thus, the amino acid residues at this position might function in the determination of product size.…”

Bacteroides thetaiotaomicron VPI‐5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions

“…The blue lines indicate the boundaries between the seven structural regions. The two catalytic amino acid residues in each sequence are indicated by the red boxes [9]. N‐VR, N‐terminal variable region; N‐CBD, N‐terminal carbohydrate‐binding region; CR‐I, conserved region I; CIT‐SR, CITase‐specific region; CR‐II, conserved region II; C‐CBD, C‐terminal carbohydrate‐binding region; C‐VR, C‐terminal variable region; BTDEX, Dex from Bacteroides thetaiotaomicron VPI‐5482 (http://www.ncbi.nlm.nih.gov/nuccore/AA078193); P7034, Dex from Paenibacillus sp.…”

Structural Elucidation of Dextran Degradation Mechanism by Streptococcus mutans Dextranase Belonging to Glycoside Hydrolase Family 66

“…598K CITase (PsCITase) (20) and Paenibacillus sp. dextranase (PsDex) (19 453 of SmDex. Biochemical analyses and structural observations agree that aspartic acid is the catalytic nucleophile and that glutamic acid is the catalytic acid/base of the GH-66 enzymes.…”

“…On the other hand, Asp 270 of CITase from Bacillus circulans T3040 (17) and Asp 243 of endodextranase from Thermotoga lettingae TMO (18), both corresponding to Asp 385 in SmDex, have been implicated as catalytic residues. Here, we recently conducted mutational analyses on two GH-66 enzymes, CITase and dextranase from Paenibacillus sp., and described three amino acid residues essential for catalysis (19,20). However, the detailed catalytic mechanism of this enzyme family has not been elucidated due to the lack of the determination of the GH-66 enzyme's three-dimensional structure.…”