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"Newton's cradle" proton relay with amide-imidic acid tautomerization in inverting cellulase visualized by neutron crystallography.

Nakamura A, Ishida T, Kusaka K, Yamada T, Fushinobu S, Tanaka I, Kaneko S, Ohta K, Tanaka H, Inaka K, Higuchi Y, Niimura N, Samejima M, Igarashi K - Sci Adv (2015)

Bottom Line: Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs).We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium.Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

ABSTRACT
Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the "Newton's cradle"-like proton relay pathway of the catalytic cycle. Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

No MeSH data available.


Proposed reaction mechanism of inverting cellulase and the close-up views of catalytic site of PcCel45A.(A) Reaction scheme of inverting cellulase. The general acid should be protonated and the general base should be deprotonated for the reaction. (B) Comparison of residues around the catalytic centers of HiCel45A (pink) and PcCel45A (green). (C) X-ray omit map of cellopentaose at subsites +1 to +5 of PcCel45A WT at room temperature (2σ level).
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Figure 1: Proposed reaction mechanism of inverting cellulase and the close-up views of catalytic site of PcCel45A.(A) Reaction scheme of inverting cellulase. The general acid should be protonated and the general base should be deprotonated for the reaction. (B) Comparison of residues around the catalytic centers of HiCel45A (pink) and PcCel45A (green). (C) X-ray omit map of cellopentaose at subsites +1 to +5 of PcCel45A WT at room temperature (2σ level).

Mentions: Hydrolase (Enzyme Commission no. 3) is the largest category of enzymes; they mediate chemical bond cleavage via the addition of a water molecule, and the catalytic residues serve to assist nucleophilic attacks of the oxygen atom of water through donating and accepting protons. Hydrolases acting on carbohydrates are called glycoside hydrolases (GHs), and they play central roles in metabolism and utilization of carbohydrates in nature. They are divided into two major groups with different hydrolytic mechanisms: retaining hydrolases afford a product with the same anomeric configuration as the substrate, whereas inverting hydrolases afford a product with the opposite anomeric configuration to the substrate (1). Both types of enzymes commonly use two acidic amino acids, typically aspartic and/or glutamic acids, at the catalytic center. Retaining glycosidases exhibit a two-step mechanism, with one of the two key catalytic residues acting as a nucleophile and the other as an acid/base; in the first step, the nucleophile attacks the anomeric center with the aid of the acidic residue to afford a glycosyl enzyme intermediate, and then the deprotonated residue serves as a base to hydrolyze the intermediate. On the other hand, in inverting enzymes, one residue serves as a general acid that protonates the glycosyl oxygen atom and the other acts as a general base that activates water to directly hydrolyze the glucosidic bond (Fig. 1A). Currently, 127 GH families are registered in the Carbohydrate Active enZymes (CAZy) database (2), and 43 of them are reported to exhibit an inverting mechanism, although the catalytic residues and mechanisms of some remain unclear (3–5).


"Newton's cradle" proton relay with amide-imidic acid tautomerization in inverting cellulase visualized by neutron crystallography.

Nakamura A, Ishida T, Kusaka K, Yamada T, Fushinobu S, Tanaka I, Kaneko S, Ohta K, Tanaka H, Inaka K, Higuchi Y, Niimura N, Samejima M, Igarashi K - Sci Adv (2015)

Proposed reaction mechanism of inverting cellulase and the close-up views of catalytic site of PcCel45A.(A) Reaction scheme of inverting cellulase. The general acid should be protonated and the general base should be deprotonated for the reaction. (B) Comparison of residues around the catalytic centers of HiCel45A (pink) and PcCel45A (green). (C) X-ray omit map of cellopentaose at subsites +1 to +5 of PcCel45A WT at room temperature (2σ level).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643802&req=5

Figure 1: Proposed reaction mechanism of inverting cellulase and the close-up views of catalytic site of PcCel45A.(A) Reaction scheme of inverting cellulase. The general acid should be protonated and the general base should be deprotonated for the reaction. (B) Comparison of residues around the catalytic centers of HiCel45A (pink) and PcCel45A (green). (C) X-ray omit map of cellopentaose at subsites +1 to +5 of PcCel45A WT at room temperature (2σ level).
Mentions: Hydrolase (Enzyme Commission no. 3) is the largest category of enzymes; they mediate chemical bond cleavage via the addition of a water molecule, and the catalytic residues serve to assist nucleophilic attacks of the oxygen atom of water through donating and accepting protons. Hydrolases acting on carbohydrates are called glycoside hydrolases (GHs), and they play central roles in metabolism and utilization of carbohydrates in nature. They are divided into two major groups with different hydrolytic mechanisms: retaining hydrolases afford a product with the same anomeric configuration as the substrate, whereas inverting hydrolases afford a product with the opposite anomeric configuration to the substrate (1). Both types of enzymes commonly use two acidic amino acids, typically aspartic and/or glutamic acids, at the catalytic center. Retaining glycosidases exhibit a two-step mechanism, with one of the two key catalytic residues acting as a nucleophile and the other as an acid/base; in the first step, the nucleophile attacks the anomeric center with the aid of the acidic residue to afford a glycosyl enzyme intermediate, and then the deprotonated residue serves as a base to hydrolyze the intermediate. On the other hand, in inverting enzymes, one residue serves as a general acid that protonates the glycosyl oxygen atom and the other acts as a general base that activates water to directly hydrolyze the glucosidic bond (Fig. 1A). Currently, 127 GH families are registered in the Carbohydrate Active enZymes (CAZy) database (2), and 43 of them are reported to exhibit an inverting mechanism, although the catalytic residues and mechanisms of some remain unclear (3–5).

Bottom Line: Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs).We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium.Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

ABSTRACT
Hydrolysis of carbohydrates is a major bioreaction in nature, catalyzed by glycoside hydrolases (GHs). We used neutron diffraction and high-resolution x-ray diffraction analyses to investigate the hydrogen bond network in inverting cellulase PcCel45A, which is an endoglucanase belonging to subfamily C of GH family 45, isolated from the basidiomycete Phanerochaete chrysosporium. Examination of the enzyme and enzyme-ligand structures indicates a key role of multiple tautomerizations of asparagine residues and peptide bonds, which are finally connected to the other catalytic residue via typical side-chain hydrogen bonds, in forming the "Newton's cradle"-like proton relay pathway of the catalytic cycle. Amide-imidic acid tautomerization of asparagine has not been taken into account in recent molecular dynamics simulations of not only cellulases but also general enzyme catalysis, and it may be necessary to reconsider our interpretation of many enzymatic reactions.

No MeSH data available.