The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase

Costa, Hernán; Distéfano, Ana Julia; Marino-Buslje, Cristina; Hidalgo, Aurélio; Berenguer, José; Bonino, Mirtha Biscoglio de Jiménez; Ferrarotti, Susana Alicia

doi:10.1007/s00253-011-3623-6

Cited by 18 publications

(12 citation statements)

References 37 publications

(43 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The residues of subsite Ϫ6 of P. macerans CGTase are highly conserved in all known CGTases. Only a few investigations on this subsite have been reported to date (15,26). One report indicated that this subsite plays a crucial role in substrate binding and affects transferase activity via an induced-fit mechanism even though it is far from the catalytic site (15).…”

Section: Construction Of Mutants By Ismmentioning

confidence: 99%

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

Han

Liu

Shin

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

d 2-O-D-Glucopyranosyl-L-ascorbic acid (AA-2G), a stable L-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from ؉2 to ؊7). In this study, iterative saturation mutagenesis (ISM) was performed on the ؊6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites-Y167, G179, G180, and N193-was first performed and revealed that four mutants-Y167S, G179R, N193R, and G180R-produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the ؊6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

show abstract

Section: Construction Of Mutants By Ismmentioning

confidence: 99%

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

Han

Liu

Shin

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…The sequences of the oligonucleotides used in this work are listed in Table I. The fragment encoding the mature CGTase without its signal peptide and with its own stop codon was cloned previously between NcoI and XhoI restriction sites of pET22b (+) expression vector to obtain plasmid pET22b(+)/CGTase (Costa et al, 2012). The purified plasmid (5 to 50 ng) was used as template for single site-directed mutagenesis reactions at positions 137, 144, 280 and 329.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…For protein overproduction, expression was carried out for 18 h with 0.4 mM IPTG. The periplasmic content of the cultures was obtained as described in Material and Methods section and its protein content was analyzed by SDS-PAGE (Costa et al, 2012). The molecular weight of the purified recombinant proteins was indistinguishable from that of the native CGTase (n-CGTase), around 74 kDa (Supplementary Figure S1).…”

Section: Selection and Production Of Mutant Cgtasesmentioning

confidence: 99%

“…Specific changes in residues involved in the catalytic site of the enzyme can affect enzymesubstrate interactions, thus driving the type of product obtained. This has been demonstrated for positions (as referred to Bacillus circulans 251 CGTase) 77 and 239 (Kelly et al, 2008), 43, 137-144 and 188 (Goh et al, 2009), 47 (Li et al, 2009a), 93 (Rostinawati et al, 2015), 89 and 372 (Li et al, 2009b), 179 (Costa et al, 2012), 195 (Xie et al, 2014a2014b), 315 (Ban et al, 2015), 577 (Huang et al, 2014;Li et al, 2016) and 100, 227 and 328 (Wang et al, 2017). Nevertheless, the mechanism determining the size of the synthesized CD remains unknown.…”

Section: Introductionmentioning

confidence: 96%

See 1 more Smart Citation

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production

Castillo

Landriel

Sánchez-Costa

et al. 2018

Protein Engineering, Design and Selection

View full text Add to dashboard Cite

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

show abstract

“…Following that study, attempts to elucidate the importance of the substrate binding subsites, particularly subsites −3, −6, and −7, were also reported [3,4]. A mutation at position 179 in subsite −6 in Bacillus circulans DF 9R CGTase both altered the CD ratio and affected catalytic efficiency [5]. By substituting the histidine with a smaller amino acid at position 43 (subsite −3) in Bacillus sp.…”

Section: Introductionmentioning

confidence: 99%

Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability

Goh

Illias

Goh

2012

IJMS

View full text Add to dashboard Cite

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

show abstract

The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase

Cited by 18 publications

References 37 publications

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production

Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability

Contact Info

Product

Resources

About