Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1

Lü, Yang; Yang, Haitao; Hu, Hai; Wang, Ying; Rao, Zihe; Jin, Cheng

doi:10.1007/s10719-008-9203-z

Cited by 25 publications

(14 citation statements)

References 27 publications

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“…Moreover, Feng et al (2005) reasoned that since deglycosylation is rate limiting, increased affinity of the acceptor would improve transglycosylation, which it is also found for a double mutant of a GH1 β -glucosidase. Higher ratios of transglycosylation have also been seen in other mutational studies with improved affinity in aglycone subsites (Champion et al 2012; Lu et al 2009; Osanjo et al 2007). …”

Section: Introductionsupporting

confidence: 55%

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

Lundemo

Karlsson

Adlercreutz

2016

Appl Microbiol Biotechnol

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Unveiling the determinants for transferase and hydrolase activity in glycoside hydrolases would allow using their vast diversity for creating novel transglycosylases, thereby unlocking an extensive toolbox for carbohydrate chemists. Three different amino acid substitutions at position 220 of a GH1 β-glucosidase from Thermotoga neapolitana caused an increase of the ratio of transglycosylation to hydrolysis (r s/r h) from 0.33 to 1.45–2.71. Further increase in r s/r h was achieved by modulation of pH of the reaction medium. The wild-type enzyme had a pH optimum for both hydrolysis and transglycosylation around 6 and reduced activity at higher pH. Interestingly, the mutants had constant transglycosylation activity over a broad pH range (5–10), while the hydrolytic activity was largely eliminated at pH 10. The results demonstrate that a combination of protein engineering and medium engineering can be used to eliminate the hydrolytic activity without affecting the transglycosylation activity of a glycoside hydrolase. The underlying factors for this success are pursued, and perturbations of the catalytic acid/base in combination with flexibility are shown to be important factors.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7833-9) contains supplementary material, which is available to authorized users.

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Section: Introductionsupporting

confidence: 55%

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

Lundemo

Karlsson

Adlercreutz

2016

Appl Microbiol Biotechnol

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“…The residue Met-226 is not directly involved in catalysis but is an important supporting residue for the catalytic activity of SpChiD. Substitution of Met-243 to Glu in family 18 chitinase from Aspergillus fumigatus showed that the mutant M243E lost the TG activity toward p-nitrophenyl-(N-acetylglucosamine) 2 but not with DP4 substrate (33). Docking studies revealed that M243E forms a hydrogen bond with backbone oxygen of Ϫ1 sugar instead of a hydrophobic stack between Met-243 and the ϩ1 subsite sugar moiety.…”

Section: Name Of the Enzymementioning

confidence: 99%

Transglycosylation by Chitinase D from Serratia proteamaculans Improved through Altered Substrate Interactions

Madhuprakash

Tanneeru

Purushotham

et al. 2012

Journal of Biological Chemistry

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Background: SpChiD, a family 18 glycosyl hydrolase, transglycosylates chitooligosaccharides. Results: Transglycosylation of SpChiD was improved in terms of the quantity of TG products produced and the extended duration of TG. Conclusion:The new findings unravel possibilities of modulating chitinases for improved transglycosylation. Significance: Variants of SpChiD can be used to develop a bio-process for large scale production of longer chain chitooligosaccharides.

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“…Family GH-18 chitinases homologous to NtChiV are known to catalyze transglycosylation reactions (Aguilera et al 2003;Lü et al 2009). Thus we examined the transglycosylation products with a more sensitive tool, MALDI-TOF-MS.…”

Section: Transglycosylationmentioning

confidence: 99%

Crystal structure and mode of action of a class V chitinase from Nicotiana tabacum

et al. 2011

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A class V chitinase from Nicotiana tabacum (NtChiV) with amino acid sequence similar to that of Serratia marcescens chitinase B (SmChiB) was expressed in E. coli and purified to homogeneity. When N-acetylglucosamine oligosaccharides [(NAG)(n)] were hydrolyzed by the purified NtChiV, the second glycosidic linkage from the non-reducing end was predominantly hydrolyzed in a manner similar to that of SmChiB. NtChiV was shown to hydrolyze partially N-acetylated chitosan non-processively, whereas SmChiB hydrolyzes the same substrate processively. The crystal structure of NtChiV was determined by the single-wavelength anomalous dispersion method at 1.2 Å resolution. The protein adopts a classical (β/α)₈-barrel fold (residues 1-233 and 303-348) with an insertion of a small (α + β) domain (residues 234-302). This is the first crystal structure of a plant class V chitinase. The crystal structure of the inactive mutant NtChiV E115Q complexed with (NAG)₄ was also solved and exhibited a linear conformation of the bound oligosaccharide occupying -2, +1, +2, and +3 subsites. The complex structure corresponds to an initial state of (NAG)₄ binding, which is proposed to be converted into a bent conformation through sliding of the +1, +2, and +3 sugar units to -1, +1, and +2 subsites. Although NtChiV is similar to SmChiB, the chitin-binding domain is present in the C-terminus of the latter, but not in the former. Aromatic amino acid residues found in the substrate binding cleft of SmChiB, including Trp97, are substituted with aliphatic residues in NtChiV. These structural differences appear to be responsible for NtChiV being a non-processive enzyme.

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Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1

Cited by 25 publications

References 27 publications

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase

Transglycosylation by Chitinase D from Serratia proteamaculans Improved through Altered Substrate Interactions

Crystal structure and mode of action of a class V chitinase from Nicotiana tabacum

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