Substrate recognition of N,N′-diacetylchitobiose deacetylase from Pyrococcus horikoshii

Nakamura, Tsutomu; Yonezawa, Yasushige; Tsuchiya, Yuko; Niiyama, Mayumi; Ida, K.; Oshima, Maki; Morita, Junji; Uegaki, Koichi

doi:10.1016/j.jsb.2016.07.015

Cited by 6 publications

(8 citation statements)

References 42 publications

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“…The results of the computational analysis of simulated site-saturation mutation indicate that F160 is an important site for the interaction with GlcNAc and has a great influence on the stability of GlcNAc binding to Dac. Moreover, as predicted in a previous study, the bond angles and spatial distances between the Tyr120 and His152 side chains support hydrogen-bonding interactions (27). Thus, we selected these five sites, R156, R157, F160, Tyr120, and His152, for saturation mutation to enhance the catalytic efficiency.…”

Section: Resultsmentioning

confidence: 61%

“…To enhance the catalytic efficiency of Dac, the molecular docking between the substrate GlcNAc and protein Dac was carried out to select the GlcNAc binding sites and determine the molecular engineering targets. As indicated in the three-dimensional (3D) structure of Dac (27), the symmetric structure, a hexamer, is made up of two trimers consisting of three identical molecules (2). According to the result of the molecular docking (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

Jiang

Niu

Liu

et al. 2019

Appl Environ Microbiol

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Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

Jiang

Niu

Liu

et al. 2019

Appl Environ Microbiol

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“…The monomeric Orf2* is self-contained, whereas in the oligomeric Pch -Dac the catalytic His259* comes from the neighboring subunit. The two deacetylases from P yrococcus horikoshii and Pyrococcus furiosus that are closely related to Pch -Dac have been described with Zn 2+ or Cd 2+ in the metal-binding site and different ligands [ 5 , 6 , 7 ]. In the complex with the reaction intermediate analog (MPG), two oxygen atoms of the ligand approach the active site but they interact asymmetrically: one makes a direct interaction with Zn 2+ (the average distance is 1.9 Å), while the other is more distant (2.8 Å).…”

Section: Resultsmentioning

confidence: 99%

“…Recent results of research on CE-14 deacetylases include crystal structures of the enzymes from Pyrococcus horikoshii and Pyrococcus furiosus [ 5 ]. The proteins have been described in complexes with an acetate ion, i.e., the product of the enzymatic reaction, with a reaction intermediate analog [ 6 ], with phosphate bound near the active site, and with Cd 2+ ions substituting Zn 2+ [ 7 ]. Here, we present an extended structural, thermodynamic and enzymatic study of a related archaeal deacetylase Pyrococcus chitonophagus .…”

Section: Introductionmentioning

confidence: 99%

Structural, Thermodynamic and Enzymatic Characterization of N,N-Diacetylchitobiose Deacetylase from Pyrococcus chitonophagus

Biniek-Antosiak

Bejger

Sliwiak

et al. 2022

IJMS

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Chitin is a major source of energy and macroelements for many organisms. An important step in its degradation is the deacetylation of chitin or its fragments. Deacetylase from the extremophile Pyrococcus chitonophagus has been analyzed by X-ray crystallography, small-angle X-ray scattering, differential scanning calorimetry, isothermal titration calorimetry and NMR to determine its structure, thermodynamics and enzymatic properties. It is a hexameric, zinc-containing metalloenzyme that retains its structural integrity up to temperatures slightly exceeding 100 °C. It removes the acetyl group specifically from the non-reducing end of the sugar substrate. Its main substrate is N,N-diacetylchitobiose but it also active, at a reduced level, toward N-acetyl-d-glucosamine or a trimer of N-acetyl-d-glucosamine units. Crystallographic analysis includes the structure of the enzyme with its main substrate approaching the active site in a monodentate manner, replacing the single water molecule that is bound at the Zn2+ cation when the ligand is absent. The Zn2+ cation remains tetrahedrally coordinated, with three of its ligands provided by the protein’s conserved His-Asp-His triad. The crystal structures are consistent with the reaction mechanism proceeding via an anhydride intermediate. Hydrolysis as the first step cannot be ruled out in a hydrated environment but no defined ‘hydrolytic water’ site can be identified in the analyzed structures.

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“…These enzymes are thermostable, with an optimal temperature of ~80 °C, which is an important requisite for industrial applications since most industrial processes are conducted under harsh conditions (e.g., high temperature and pressure). Previous determination of the chemical structures of ChiA and Dac provided insights into their catalytic mechanism and adaptation to extremely high temperatures [4,5,6,7,8,9,10]. However, for almost 14 years after the first description of GlmA, its structure has remained unknown.…”

Section: Introductionmentioning

confidence: 99%

Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase

Mine

Watanabe²

2019

IJMS

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The archaeal exo-β-d-glucosaminidase (GlmA), a thermostable enzyme belonging to the glycosidase hydrolase (GH) 35 family, hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a novel enzyme in terms of its primary structure, as it is homologous to both GH35 and GH42 β-galactosidases. The catalytic mechanism of GlmA is not known. Here, we summarize the recent reports on the crystallographic analysis of GlmA. GlmA is a homodimer, with each subunit comprising three distinct domains: a catalytic TIM-barrel domain, an α/β domain, and a β1 domain. Surprisingly, the structure of GlmA presents features common to GH35 and GH42 β-galactosidases, with the domain organization resembling that of GH42 β-galactosidases and the active-site architecture resembling that of GH35 β-galactosidases. Additionally, the GlmA structure also provides critical information about its catalytic mechanism, in particular, on how the enzyme can recognize glucosamine. Finally, we postulate an evolutionary pathway based on the structure of an ancestor GlmA to extant GH35 and GH42 β-galactosidases.

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Substrate recognition of N,N′-diacetylchitobiose deacetylase from Pyrococcus horikoshii

Cited by 6 publications

References 42 publications

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

Structural, Thermodynamic and Enzymatic Characterization of N,N-Diacetylchitobiose Deacetylase from Pyrococcus chitonophagus

Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase

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