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

Abstract

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. #

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…Classically, specificity engineering modified the product composition for cyclodextrin glucanotransferases, neopullulanases, and maltogenic α-amylase, by taking advantage of insight into the conserved sequence motifs extending at four active site β-strands (Kuriki et al 1996;Beier et al 2000;MacGregor et al 2001;Leemhuis et al 2003). Recently, engineering of GH70 members as guided by sequences of enzymes with assigned product bond-type specificity by multiple mutational substitutions in conserved sequence motifs of GH70 succeeded to alter the α-glucan product bonds of reuteransucrase to be mainly of α-1,6-rather than α-1,4-glucosidic linkage specificity (Kralj et al 2005).…”

Mutations affecting the activity of the cyclodextrin glucanotransferase of Paenibacillus pabuli US132: insights into the low hydrolytic activity of cyclodextrin glucanotransferases

“…2). This loop was described as the most significant structural feature related to the differences in the product specificities of Novamyl and CGTases (Dauter et al 1999;Leemhuis et al 2003b). In fact, the presence of the loop at the donor subsites −3/ − 4 could sterically hinder CDs formation and make small oligosaccharides in an optimum orientation for hydrolysis.…”

“…The study also involved the addition of the G180D mutation since a side chain at position 180 strongly interferes with the selection of longer linear substrates as well as the circularization process, which explains the conservation of Gly180 in all CGTases (Leemhuis et al 2002b). Moreover, the equivalent aspartate in Novamyl is known to form a hydrogen bond with the serine in the loop PAGFS, which may be important for the stabilization of the insertion conformation (Leemhuis et al 2003b). …”

“…Based on a rational protein engineering approach, Novamyl was previously converted from a maltogenic amylase into a CD-forming enzyme through the introduction of few mutations (Beier et al 2000). Conversely, the Tabium CGTase was converted into a starch hydrolase using the opposite experiment (Leemhuis et al 2003b). However, it is worth noting that the latter CGTase represents a particular case since it exhibited the highest initial hydrolytic activity ever recorded for a CGTase.…”

Improved activity of β-cyclodextrin glycosyltransferase from Bacillus sp. N-227 via mutagenesis of the conserved residues

“…alkalophilus enhanced the cyclization activity and decreased the hydrolytic activity of bCGTase (Hyun-Dong et al 2000). Contrarily, mutant A230V CGTase in Bacillus circulans strain 251 improved the hydrolytic activity but reduced cyclization activity (Leemhuis et al 2003a). Additionally, Dijkhuizen and collages uncovered that mutation of tyrosine 195, which located in the central active site cleft of CGTase, significantly suppressed the cyclodextrin formation (Penninga et al 1995).…”