The hydrogen-bond network around Glu160 contributes to the structural stability of chitosanase CsnA from Renibacterium sp. QD1

Han, Yujuan; Yu, Rilei; Gao, Peixin; Lu, Xin; Yu, Wengong

doi:10.1016/j.ijbiomac.2017.11.071

Cited by 15 publications

(13 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The mutation of the hydrophilic Asp229 or Gln261 to small hydrophobic Ala led to the disruption of the hydrogen bond networks involving these two residues and their surrounding residues in Chi23, and therefore the protein structure of Chi23 became less rigid and less stable at high temperatures. The contribution of hydrogen bonds to protein thermostability was also reported in other glycoside hydrolases (Vogt et al, 1997; Han et al, 2018). In addition, the absence of long flexible loops may also benefit the high thermostability of Chi23.…”

Section: Resultssupporting

confidence: 66%

Structural Insight Into Chitin Degradation and Thermostability of a Novel Endochitinase From the Glycoside Hydrolase Family 18

et al. 2019

View full text Add to dashboard Cite

Bacterial endochitinases play important roles in environmental chitin degradation and have good applications. Although the structures of some endochitinases, most belonging to the glycoside hydrolase (GH) family 18 and thermostable, have been reported, the structural basis of these enzymes for chitin degradation still remain unclear due to the lack of functional confirmation, and the molecular mechanism for their thermostability is also unknown. Here, we characterized a GH18 endochitinase, Chi23, from marine bacterium Pseudoalteromonas aurantia DSM6057, and solved its structure. Chi23 is a thermostable enzyme that can non-processively hydrolyze crystalline and colloidal chitin. Chi23 contains only a catalytic domain that adopts a classical (β/α)8 TIM-barrel fold. Compared to other GH18 bacterial endochitinases, Chi23 lacks the chitin-binding domain and the β-hairpin subdomain, indicating that Chi23 has a novel structure. Based on structural analysis of Chi23 docked with (GlcNAc)5 and mutational analysis, the key catalytic residue (Glu117) and seven substrate-binding residues (Asn9, Gln157, Tyr189, Asn190, Asp229, Trp260, and Gln261) are revealed. Among these identified residues, Asn9, Asp229 and Gln261 are unique to Chi23, and their cumulative roles contribute to the activity of Chi23 against both crystalline and soluble chitin. Five substrate-binding residues (Tyr189, Asn190, Asp229, Trp260, and Gln261) are found to play important roles in maintaining the thermostability of Chi23. In particular, hydrogen bond networks involving Asp229 and Gln261 are formed to stabilize the protein structure of Chi23. Phylogenetic analysis indicated that Chi23 and its homologs represent a new group of GH18 endochitinases, which are widely distributed in bacteria. This study sheds light on the molecular mechanism of a GH18 endochitinase for chitin degradation.

show abstract

Section: Resultssupporting

confidence: 66%

Structural Insight Into Chitin Degradation and Thermostability of a Novel Endochitinase From the Glycoside Hydrolase Family 18

et al. 2019

View full text Add to dashboard Cite

show abstract

“…QD1. 13 Sheng et al improved the thermostability on chitosanase from Bacillus ehimensis by introducing disulfide bonds. 14 As described above, the catalytic hydrolysis mechanism of GH46 chitosanases has been well illustrated.…”

Section: ■ Introductionmentioning

confidence: 94%

Improvement of the Catalytic Activity of Chitosanase BsCsn46A from Bacillus subtilis by Site-Saturation Mutagenesis of Proline121

Guo

Wang

Zhang

et al. 2021

J. Agric. Food Chem.

View full text Add to dashboard Cite

BsCsn46A, a GH46 family chitosanase from Bacillus subtilis, has great potential for industrial chitooligosaccharide production due to its high activity and stability. In this study, a special amino acid Pro121 was identified not fit in the helix structure, which was located in the opposite side of the active center in BsCsn46A, by the PoPMuSiC algorithm. Then, saturation mutagenesis was performed to explore the role of the site amino acid 121. Compared with the wild type, the specific activity of P121N, P121C, and P121V was increased by 1.69-, 1.97-, and 2.15-fold, respectively. In particular, the specific activity of P121N was increased without loss of thermostability, indicating that replacing the structural stiffness of proline in the helical structure could significantly improve the chitosanase activity. The K m values of P121N, P121C, and P121V decreased significantly, indicating that the affinity between the enzyme–substrate complex was enhanced. Through molecular docking, it was found that the increase of hydrogen bonds and van der Waals force between the enzyme–substrate complex and the removal of unfavorable bonds might be the main reason for the change of enzyme properties. In addition, the optimal temperature of the three mutants changed from 60 to 55 °C. These results indicate that the site 121 plays a critical role in the catalytic activity and enzymatic properties of chitosanase. To our knowledge, the results provide novel data on chitosanase activity and identify an excellent candidate of industrial chitosanase.

show abstract

“…(f)]. An increase in the number of hydrogen bonds has been suggested as one of the principal determinants in enhancing thermostability . A decrease would be expected to cause protein instability.…”

Section: Resultsmentioning

confidence: 99%

“…An increase in the number of hydrogen bonds has been suggested as one of the principal determinants in enhancing thermostability. 19 A decrease would be expected to cause protein instability. By combining the kinetic parameters and thermodynamic activation parameters in Table 2, S171A might be seen to have slightly reduced stability compared to the WT, indicating a small alteration in its 3D structure.…”

Section: Molecular Docking Analysismentioning

confidence: 99%

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

Mei

et al. 2018

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

BACKGROUND Myofibril‐bound serine proteinase (MBSP) comprises a class of trypsin‐type serine proteinases that can specifically cleave the carboxyl side of peptide bonds of arginine or lysine residues. To obtain higher specificity MBSPs, mutants were designed based on the substrate‐binding pocket and constructed through site‐directed mutagenesis. RESULTS The wild‐type (WT) and mutants were expressed in Pichia pastoris, and its enzymatic properties indicated that the mutants D170S, D170T, D170K and D170T/G203A were not biologically active. However, S171A specifically cleaved arginine residues and G203A preferably hydrolyzed lysine residues. The secondary structures of mutants were approximately similar to that of the WT according to the results of circular dichroism spectroscopy. Additionally, molecular docking showed that substrates were able to combine with S171A within the critical areas through hydrophobic interaction, hydrogen bonds and van der Waals electrostatic force. CONCLUSION The structural stability of MBSP was consistent using amino acid substitution. Due to the interaction pattern of substrates and mutant enzyme changes, only S171A was selective in cutting Arg residues, which illustrates how the catalytic triad and substrate‐binding pocket of MBSP plays an important role during enzymatic hydrolysis of substrates. © 2018 Society of Chemical Industry

show abstract

The hydrogen-bond network around Glu160 contributes to the structural stability of chitosanase CsnA from Renibacterium sp. QD1

Cited by 15 publications

References 33 publications

Structural Insight Into Chitin Degradation and Thermostability of a Novel Endochitinase From the Glycoside Hydrolase Family 18

Structural Insight Into Chitin Degradation and Thermostability of a Novel Endochitinase From the Glycoside Hydrolase Family 18

Improvement of the Catalytic Activity of Chitosanase BsCsn46A from Bacillus subtilis by Site-Saturation Mutagenesis of Proline121

Altering the substrate specificity of a myofibril‐bound serine proteinase from Crucian carp by site‐directed mutagenesis

Contact Info

Product

Resources

About