Effect on product specificity of cyclodextrin glycosyltransferase by site‐directed mutagenesis

Kim, Young Ho; Bae, Kwang‐Hee; Kim, Tae Jip; Park, Kwan Hwa; Lee, Hyun Soo; Byun, Sun‐Sig

doi:10.1080/15216549700201231

Cited by 16 publications

(24 citation statements)

References 3 publications

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“…Nevertheless, the mutants A and D retained a small cyclodextrin forming activity, whereas the G2-amylase has no cyclization activity, suggesting that the conformation of the five-residue insertion is different in Tabium CGTase mutants A and D. However, other differences between the two (mutant) enzymes may also contribute to this different reaction specificity. A simulation of the cyclization reaction catalyzed by B. circulans strain 251 CGTase has revealed that nearly all substrate binding subsites of CGTase contribute to the cyclization reaction , which is in agreement with mutation studies (van der Veen et al, 2000b;Leemhuis et al, 2002c;Parsiegla et al, 1998;Kim et al, 1997;Nakamura et al, 1993Nakamura et al, , 1994a. Thus, the limited cyclization activity of the five-residue insertion mutants might be explained by the functionality of the acceptor binding subsites of CGTase, which are still specialized to form cyclodextrins, even though the interactions at the remote donor subsites are not possible in these mutants.…”

Section: Increased Ratio Of Hydrolysis/cyclization Reaction Specificisupporting

confidence: 59%

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Leemhuis

Kragh²,

Dijkstra

et al. 2003

Journal of Biotechnology

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Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exospecificity Leemhuis, H; Kragh, KM; Dijkstra, BW; Dijkhuizen, Lubbert; Kragh, Karsten M.; Dijkstra, Bauke W. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. AbstractCyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic a-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite '/3 had a different conformation, providing the G2-amylase with more space at acceptor subsite '/3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites (/3/(/4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite '/3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites. #

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Section: Increased Ratio Of Hydrolysis/cyclization Reaction Specificisupporting

confidence: 59%

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Leemhuis

Kragh²,

Dijkstra

et al. 2003

Journal of Biotechnology

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“…Subsite Ϫ3 mutations in the alkalophilic Bacillus sp. I-5 CGTase also changed the product specificity [68].…”

Section: Protein Engineering Of the Product Specificity Of Cgtasementioning

confidence: 99%

Catalytic mechanism and product specificity of cyclodextrin glycosyltransferase, a prototypical transglycosylase from the α-amylase family

Uitdehaag

Veen

Dijkhuizen

et al. 2002

Enzyme and Microbial Technology

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Catalytic mechanism and product specificity of cyclodextrin glycosyltransferase, a prototypical transglycosylase from the α-amylase family Uitdehaag, Joost C.M.; Veen, Bart A. van der; Dijkhuizen, Lubbert; Dijkstra, Bauke W. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. AbstractThe catalytic mechanism of cyclodextrin glycosyltransferase, a member of the ␣-amylase family, is reviewed. The focus is put on the bond cleavage mechanism, the nature of the transition state and of the covalent intermediate, and on the stereo-electronic and lateral protonation contributions to catalysis. The functions in catalysis of the absolutely conserved residues in this family are discussed. Finally, the fascinating capability of cyclodextrin glycosyltransferase to produce cyclodextrins from linear starch oligosaccharide chains is reviewed, together with protein engineering studies to modify the enzyme's product specificity.

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“…This observation was in agreement with Y100F substitution in Bacillus sp. I-5 CGTase (93 in BA2-5a) did not change its K m (Kim et al, 1997) . Using the molecular docking approach, there was no interaction type change of Phe93 with substrate observed in Y93F CGTase compared to Tyr93 in wild type.…”

Section: Kinetic Parameters Of Y93fmentioning

confidence: 99%

“…The values of K m and k cat were determined by the nonlinear least-squares method with Hill-equation (Kim et al, 1997).…”

Section: Assay Of Protein Thermal Shiftmentioning

confidence: 99%

“…I-5 CGTase, the amino acid residue at position 100 is located near the putative active centre, substitution at position 100 (Y100F) had yielded more product ratio of β-CD. The variations in product specificity between the various CGTases can be ascribed to the relationship between their similar catalytic centre and different subsite structure (Kim et al, 1997). Therefore, mutant BA2-5a CGTase was designed by site-directed mutagenesis which would result in Y93F (100 in Bacillus sp.…”

Section: Introductionmentioning

confidence: 99%

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Y93F Substitution of Cyclodextrin Glucanotransferase from Bacillus sp. A2-5a and its Enzyme Characterization

Rostinawat¹,

Riani²,

Elfahmi³

et al. 2015

Biotechnology

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A B S T R A C TIn this recent study, Tyr93 of Bacillus sp. A2-5a (BA2-5a) cyclodextrin glucanotransferase (CGTase) was replaced with Phe residue by site-directed mutagenesis to study its role on the product specificity and kinetic properties. Molecular docking approach was also applied to acquire a detailed analysis of enzyme-substrate interaction. The Y93F was overproduced in Escherichia coli BL21 (DE3) and purified by Ni-NTA resin. The purified Y93F CGTase was 76.39 kDa based on 10% SDS-PAGE analysis and showed both β-cyclization and starch hydrolysis activities in a zymography assay. There was no major structural conformational change in the Y93F since, its optimum and stability of temperature and pH were the same as the wild type. The substitution did not alter the cooperativity property of the enzyme. Our work provided the new evidence that Y93F substitution caused no α-cyclodextrin (CD) formation in BA2-5a CGTase. In addition, the Y93F produced more β-CD and decreased its k cat and k cat /K m . In conclusion, Y93F substitution of BA2-5a CGTase alters the enzyme kinetic property and product specificity.

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Effect on product specificity of cyclodextrin glycosyltransferase by site‐directed mutagenesis

Cited by 16 publications

References 3 publications

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Catalytic mechanism and product specificity of cyclodextrin glycosyltransferase, a prototypical transglycosylase from the α-amylase family

Y93F Substitution of Cyclodextrin Glucanotransferase from Bacillus sp. A2-5a and its Enzyme Characterization

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