Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01

Lyu, Qianqian; Shi, Yanhong; Wang, Song; Yang, Yan; Han, Baoqin; Liu, Wanshun; Jones, David N. M.; Liu, Weizhi

doi:10.1016/j.bbagen.2015.06.011

Cited by 38 publications

(35 citation statements)

References 44 publications

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“…A comparison of the chitosan-free and chitosan-bound forms of CsnOU01 reveals that the enzyme must undergo the opening conformational transition to allow for the chitosan polymer binding, while the protein structure must close upon the substrate for catalysis and open again for the product release (Lyu et al 2015). A similar conformational change was also reported in a family GH19 chitinase from rye seeds (Ohnuma et al 2013), indicating a close relationship between the mechanisms of GH46 and GH19 enzymes .…”

Section: Introductionmentioning

confidence: 75%

“…A number of the assigned NH cross-peaks, including N23, W28, A30, G39, G43, G47, G153, T157, and D232, were significantly perturbed by addition of chitosan oligomers. Figure 2 shows the backbone superimposition of the substrate-bound closed conformation of CsnOU01 (Lyu et al 2015) with the ligand-free open form of CsnN174 . Residues with resolved NH cross-peaks sensitive to ligand binding are indicated on both closed and open structures to highlight their localization to the important functional regions of the enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…3). It is unlikely, that two substrate molecules may be bound at the same time to chitosanase because the long-chain oligosaccharide such as (GlcN)6 is known to fully engage the binding cleft (Lyu et al 2015)-eliminating two-binding site mechanism Fig. 3c.…”

Section: Resultsmentioning

confidence: 99%

“…2) along with kinetic and thermodynamic studies led to the proposed induced-fit interaction mechanism (Lyu et al 2015). Site-resolved analysis of chemical shift perturbations may be informative on relative roles of different residues in separate steps of enzyme-substrate interactions.…”

Section: Discussionmentioning

confidence: 99%

“…(CsnOU01), whose amino acid sequence is 60% homologous to that of CsnN174, was solved by X-ray crystallography in a complex with chitosan hexamer (GlcN)6 (Lyu et al 2015). The two chitosanases were found to have a similar fold, and the complex structure revealed interaction of (GlcN)6 with the enzyme binding cleft highlighting the dominant role of the −2, −1 and +1 subsites in substrate binding and catalysis.…”

Section: Introductionmentioning

confidence: 99%

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NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

et al. 2017

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Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.

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Section: Introductionmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

et al. 2017

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Crystal structure of a family 80 chitosanase from Mitsuaria chitosanitabida

et al. 2017

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Chitosanases belong to glycoside hydrolase families 5, 7, 8, 46, 75 and 80 and hydrolyse glucosamine polymers produced by partial or full deacetylation of chitin. Herein, we determined the crystal structure of chitosanase from the β-proteobacterium, Mitsuaria chitosanitabida, (McChoA) at 1.75 Å resolution; the first structure of a family 80 chitosanase. McChoA is a 34 kDa extracellular protein of 301 amino acids that fold into two (upper and lower) globular domains with an active site cleft between them. Key substrate-binding features are conserved with family 24 lysozymes and family 46 chitosanases. The distance between catalytic residues E41 and E61 (10.8 Å) indicates an inverting type mechanism. Uniquely, three disulphide bridges and the C terminus might contribute to enzyme activity.

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Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Regel

Weikert

Niehues

et al. 2018

Biotech & Bioengineering

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Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (-2), which is impossible for the wildtype enzyme.

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Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01

Cited by 38 publications

References 44 publications

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

NMR line shape analysis of a multi-state ligand binding mechanism in chitosanase

Crystal structure of a family 80 chitosanase from Mitsuaria chitosanitabida

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

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