Four Aromatic Residues in the Active Center of Cyclodextrin Glucanotransferase from Alkalophilic Bacillus sp. 1011: Effects of Replacements on Substrate Binding and Cyclization Characteristics

Nakamura, Akira; Haga, Keiko; Yamane, Kunio

doi:10.1021/bi00199a015

Cited by 71 publications

(64 citation statements)

References 55 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: 79%

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: 79%

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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“…Therefore, this mutant could not form an inclusion complex with a spiral substrate and hindered the formation of ␣-cyclodextrin (31). Meanwhile, due to the possible decrease in hydrogen bonding with the substrate sugars, the ability of the mutant Y195S to bind cyclodextrin decreased, while the effect of linear oligosaccharide was unapparent (31). However, when the Ser195 participated in action with either Arg260 or Lys265 or both of them, as shown in Fig.…”

Section: Resultsmentioning

confidence: 98%

Systems Engineering of Tyrosine 195, Tyrosine 260, and Glutamine 265 in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Enhance Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

Han

Liu

Shin

et al. 2013

Appl Environ Microbiol

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In this work, the site saturation mutagenesis of tyrosine 195, tyrosine 260 and glutamine 265 in the cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase for maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the synthesis of 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G). Specifically, the site-saturation mutagenesis of three sites-tyrosine 195, tyrosine 260, and glutamine 265-was performed, and it was found that the resulting mutants (containing the mutations Y195S [tyrosine ¡ serine], Y260R [tyrosine ¡ arginine], and Q265K [glutamine ¡ lysine]) produced higher AA-2G yields than the wild type and the other mutant CGTases when maltodextrin was used as the glycosyl donor. Furthermore, double and triple mutations were introduced, and four mutants (containing Y195S/ Y260R, Y195S/Q265K, Y260R/Q265K, and Y260R/Q265K/Y195S) were obtained and evaluated for the capacity to produce AA-2G. The Y260R/Q265K/Y195S triple mutant produced the highest titer of AA-2G at 1.92 g/liter, which was 60% higher than that (1.20 g/liter) produced by the wild-type CGTase. The kinetics analysis of AA-2G synthesis by the mutant CGTases confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, all seven mutants had lower cyclization activities and higher hydrolysis and disproportionation activities. Finally, the mechanism responsible for the enhanced substrate specificity was explored by structure modeling, which indicated that the enhancement of maltodextrin specificity may be related to the changes of hydrogen bonding interactions between the side chain of residue at the three positions (195, 260, and 265) and the substrate sugars. This work adds to our understanding of the synthesis of AA-2G and makes the Y260R/ Q265K/Y195S mutant a good starting point for further development by protein engineering.

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“…Alignment of the available amylomaltase and D-enzyme sequences also indicated that Y54 is not conserved in this enzyme family. Alterations in reaction specificity after amino acid substitution have been reported for CGTase (8,11,12,22,23,24,25). Nakamura et al (11) introduced mutations into four aromatic residues, F183, Y195, F259, and F283, located in the center of the active cleft of CGTase from alkalophilic Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

Fujii

Minagawa

Terada

et al. 2005

Appl Environ Microbiol

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Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of ␣-1,4 glucans to produce cyclic ␣-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.

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Four Aromatic Residues in the Active Center of Cyclodextrin Glucanotransferase from Alkalophilic Bacillus sp. 1011: Effects of Replacements on Substrate Binding and Cyclization Characteristics

Cited by 71 publications

References 55 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

Systems Engineering of Tyrosine 195, Tyrosine 260, and Glutamine 265 in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Enhance Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

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