Molecular engineering of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040: structural determinants for the reaction product size and reactivity

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.

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Crystal structures of the GH6 Orpinomyces sp. Y102 CelC7 enzyme with exo and endo activity and its complex with cellobiose

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319013597 ◽

2019 ◽

Vol 75 (12) ◽

pp. 1138-1147

Author(s):

Hsiao-Chuan Huang ◽

Liu-Hong Qi ◽

Yo-Chia Chen ◽

Li-Chu Tsai

Keyword(s):

Enzymatic Hydrolysis ◽

Crystal Structures ◽

Active Site ◽

Binding Sites ◽

Glycoside Hydrolase ◽

Catalytic Domain ◽

Glycoside Hydrolase Family ◽

Substrate Binding Sites ◽

Key Residues ◽

Hydrolase Family

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose-bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate-binding sites have both tunnel-like and open-cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC-MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7.

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Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides

Biotechnology Letters ◽

10.1007/s10529-007-9627-8 ◽

2007 ◽

Vol 30 (5) ◽

pp. 869-875 ◽

Cited By ~ 10

Author(s):

Guangfei Liu ◽

Jiti Zhou ◽

Jing Wang ◽

Bin Yan ◽

Jingmei Li ◽

...

Keyword(s):

Rhodobacter Sphaeroides ◽

Binding Sites ◽

Substrate Binding ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Substrate Binding Sites

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Aromatic residues within the substrate-binding cleft of Bacillus circulans chitinase A1 are essential for hydrolysis of crystalline chitin

Biochemical Journal ◽

10.1042/bj20030419 ◽

2003 ◽

Vol 376 (1) ◽

pp. 237-244 ◽

Cited By ~ 84

Author(s):

Takeshi WATANABE ◽

Yumiko ARIGA ◽

Urara SATO ◽

Tadayuki TORATANI ◽

Masayuki HASHIMOTO ◽

...

Keyword(s):

Catalytic Domain ◽

Substrate Binding ◽

Substrate Inhibition ◽

Site Directed Mutagenesis ◽

Bacillus Circulans ◽

Aromatic Residues ◽

Km Value ◽

Chitin Hydrolysis ◽

Binding Cleft ◽

Hydrolysis Of

Bacillus circulans chitinase A1 (ChiA1) has a deep substrate-binding cleft on top of its (β/α)8-barrel catalytic domain and an interaction between the aromatic residues in this cleft and bound oligosaccharide has been suggested. To study the roles of these aromatic residues, especially in crystalline-chitin hydrolysis, site-directed mutagenesis of these residues was carried out. Y56A and W53A mutations at subsites −5 and −3, respectively, selectively decreased the hydrolysing activity against highly crystalline β-chitin. W164A and W285A mutations at subsites +1 and +2, respectively, decreased the hydrolysing activity against crystalline β-chitin and colloidal chitin, but enhanced the activities against soluble substrates. These mutations increased the Km-value when reduced (GlcNAc)5 (where GlcNAc is N-acetylglucosamine) was used as the substrate, but decreased substrate inhibition observed with wild-type ChiA1 at higher concentrations of this substrate. In contrast with the selective effect of the other mutations, mutations of W433 and Y279 at subsite −1 decreased the hydrolysing activity drastically against all substrates and reduced the kcat-value, measured with 4-methylumbelliferyl chitotrioside to 0.022% and 0.59% respectively. From these observations, it was concluded that residues Y56 and W53 are only essential for crystalline-chitin hydrolysis. W164 and W285 are very important for crystalline-chitin hydrolysis and also participate in hydrolysis of other substrates. W433 and Y279 are both essential for catalytic reaction as predicted from the structure.

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