Mutation of a Conserved Tryptophan in the Chitin-Binding Cleft of<i>Serratia marcescens</i>Chitinase A Enhances Transglycosylation

Aronson, Nathan N.; Halloran, Brian; Alexeyev, Mikhail; Zhou, Xiang; Wang, Yujun; Meehan, Edward J.; Chen, Liqing

doi:10.1271/bbb.70.243

Cited by 55 publications

(51 citation statements)

References 30 publications

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“…MD Simulation Protocol-Crystal structures from the PDB were used to build ChiA (20), ChiB (34), and 3IAN, whereas the structure presented in this work was used to build ChiC2. For ChiC2 and 3IAN, the chitin ligand (GlcNAc 7 and GlcNAc 6 , respectively) spanning the substrate-binding cleft was modeled from an alignment of a combination of ligands bound in structurally similar enzymes (49,50).…”

Section: Methodsmentioning

confidence: 99%

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Payne

Baban

Horn

et al. 2012

Journal of Biological Chemistry

112

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Background: Nature employs processive and nonprocessive glycoside hydrolases to degrade polysaccharides. Results: We solved the Serratia marcescens nonprocessive chitinase (ChiC2) structure and used simulation to identify dynamic hallmarks of processivity in S. marcescens chitinases. Conclusion: Dynamic metrics complement structural insights in determining processivity. Significance: Identification of hallmarks of processivity is a key step toward development of a general, molecular-level theory of glycoside hydrolase processivity.

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Section: Methodsmentioning

confidence: 99%

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Payne

Baban

Horn

et al. 2012

Journal of Biological Chemistry

112

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“…11) Hydrolysis reactions were done as previously reported, 12,13) and products were analyzed by an isocratic HPLC method with a TSK-Gel Amide-80 column from TosoHaas (Montgomeryville, PA) that allowed determination of both -andanomers.…”

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confidence: 99%

“…Immediately after filtration to separate out the enzyme, the sizes and anomers of products were determined by HPLC (B). [12][13][14] The -anomer of each chitooligosaccharide (N2-N5) and GlcNAc (N1) has a shorter retention time than their -anomer. One half of the reaction filtrate was post-incubated 90 min at 37 C (C) to equilibrate anomers.…”

mentioning

confidence: 99%

“…Total substrate disappearance was analyzed by HPLC. [12][13][14] (GlcNAc)2 ( ), (GlcNAc)3 ( ), (GlcNAc)4 ( ), (GlcNAc)5 ( ), and (GlcNAc)6 ( ). The dashed line through the data points estimates an equivalent disappearance rate for all substrates except (GlcNAc)2 ( ).…”

mentioning

confidence: 99%

“…Products were analyzed by HPLC. [12][13][14] -(GlcNAc)3 ( ), -(GlcNAc)3 ( ), -D-GlcNAc ( ), and -(GlcNAc)2 ( ).…”

mentioning

confidence: 99%

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Optimum Substrate Size and Specific Anomer Requirements for the Reducing-End Glycoside Hydrolase Di-N-Acetylchitobiase

Aronson

Halloran

2006

Bioscience, Biotechnology, and Biochemistry

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Hypertransglycosylating Variants of the GH20 β‐N‐Acetylhexosaminidase for the Synthesis of Chitooligomers

et al. 2022

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Fungal β-N-acetylhexosaminidases of the CAZy family 20 of glycoside hydrolases are wellestablished tools for the enzymatic synthesis of a wide variety of natural and modified oligosaccharides and glycoconjugates. In order to increase their synthetic efficiency, the β-N-acetylhexosaminidase from Aspergillus oryzae (AoHex) was employed as a model enzyme for enzyme engineering aiming at shifting the reaction course from hydrolysis toward transglycosylation. Specifically, nine mutant variants of AoHex were designed by molecular modeling based on its crystal structure and molecular dynamics simulations. The selected mutation hotspots included the tyrosine residue at the active site, which stabilizes the transition state of the reaction, and two residues at the aglycone-binding site, which were replaced by tryptophan residues to increase the hydrophobicity of this subsite. Besides the individual mutants, combined double-mutant variants were also prepared and characterized. As a result, eight out of the studied new AoHex variants had transglycosidase activity, with V306W/Y445N AoHex being a superior transglycosidase with a transglycosylation-to-hydrolysis ratio greater than 110, which is entirely unique among the hypertransglycosylating glycosidase mutants including the GH20 β-N-acetylhexosaminidases.

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Mutation of a Conserved Tryptophan in the Chitin-Binding Cleft ofSerratia marcescensChitinase A Enhances Transglycosylation

Cited by 55 publications

References 30 publications

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Optimum Substrate Size and Specific Anomer Requirements for the Reducing-End Glycoside Hydrolase Di-N-Acetylchitobiase

Hypertransglycosylating Variants of the GH20 β‐N‐Acetylhexosaminidase for the Synthesis of Chitooligomers

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