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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.


Detailed analysis of protonation state of Asn92 by x-ray and neutron crystallographies.(A) Determination of orientation and form of Asn92 at room temperature by x-ray diffraction. B-factor values (Å2) are shown above atoms (2Fobs − Fcalc map: 1.5σ in blue, Fobs − Fcalc map: 3.0σ in green and red for positive and negative). (B) Determination of orientation and protonation/deuteration of Asn92 at room temperature by neutron diffraction. B-factor values (Å2) of oxygen and nitrogen atoms are shown above atoms. Ratios of H/D are also shown (2Fobs − Fcalc map: 1.0σ in purple, Fobs − Fcalc map: 2.0σ in green and red for positive and negative).
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Figure 2: Detailed analysis of protonation state of Asn92 by x-ray and neutron crystallographies.(A) Determination of orientation and form of Asn92 at room temperature by x-ray diffraction. B-factor values (Å2) are shown above atoms (2Fobs − Fcalc map: 1.5σ in blue, Fobs − Fcalc map: 3.0σ in green and red for positive and negative). (B) Determination of orientation and protonation/deuteration of Asn92 at room temperature by neutron diffraction. B-factor values (Å2) of oxygen and nitrogen atoms are shown above atoms. Ratios of H/D are also shown (2Fobs − Fcalc map: 1.0σ in purple, Fobs − Fcalc map: 2.0σ in green and red for positive and negative).

Mentions: The orientation and protonation/deuteration state of Asn92 were next examined by x-ray (Fig. 2A, a to d) and neutron (Fig. 2B, a to d) crystallography. When the oxygen atom was facing the catalytic center, a strongly positive Fobs − Fcalc map was observed around the nitrogen atom in the x-ray analysis (Fig. 2A, a). In contrast, positive and negative Fobs − Fcalc maps were observed at the top and bottom of the oxygen atom, respectively, when the nitrogen atom was facing the catalytic center (Fig. 2A, b). The imidic acid form of Asn92, where the nitrogen atom faces the catalytic center, showed the weakest Fobs − Fcalc map in the x-ray analysis (Fig. 2A, d). In the neutron structure, strongly positive Fobs − Fcalc maps, which reflect deuteration/protonation of oxygen, were observed around the oxygen atom in the amide form of Asn92 (Fig. 2B, a and b). The hydrogen/deuterium (H/D) ratio was 0.3:0.7 under the crystallization conditions because of the difficulty of preparing fully deuterated 3MPD, and the H/D ratio of the imidic acid form of Asn92 is consistent with that value (Fig. 2B, d). The combined x-ray and neutron crystallography results indicate that the side chain of the asparagine residue takes the imidic acid form (HO–CX=NH), but not the typical amide form (O=CX–NH2), providing the first direct evidence that the imidic acid plays a key role in the hydrolysis.


"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)

Detailed analysis of protonation state of Asn92 by x-ray and neutron crystallographies.(A) Determination of orientation and form of Asn92 at room temperature by x-ray diffraction. B-factor values (Å2) are shown above atoms (2Fobs − Fcalc map: 1.5σ in blue, Fobs − Fcalc map: 3.0σ in green and red for positive and negative). (B) Determination of orientation and protonation/deuteration of Asn92 at room temperature by neutron diffraction. B-factor values (Å2) of oxygen and nitrogen atoms are shown above atoms. Ratios of H/D are also shown (2Fobs − Fcalc map: 1.0σ in purple, Fobs − Fcalc map: 2.0σ in green and red for positive and negative).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Detailed analysis of protonation state of Asn92 by x-ray and neutron crystallographies.(A) Determination of orientation and form of Asn92 at room temperature by x-ray diffraction. B-factor values (Å2) are shown above atoms (2Fobs − Fcalc map: 1.5σ in blue, Fobs − Fcalc map: 3.0σ in green and red for positive and negative). (B) Determination of orientation and protonation/deuteration of Asn92 at room temperature by neutron diffraction. B-factor values (Å2) of oxygen and nitrogen atoms are shown above atoms. Ratios of H/D are also shown (2Fobs − Fcalc map: 1.0σ in purple, Fobs − Fcalc map: 2.0σ in green and red for positive and negative).
Mentions: The orientation and protonation/deuteration state of Asn92 were next examined by x-ray (Fig. 2A, a to d) and neutron (Fig. 2B, a to d) crystallography. When the oxygen atom was facing the catalytic center, a strongly positive Fobs − Fcalc map was observed around the nitrogen atom in the x-ray analysis (Fig. 2A, a). In contrast, positive and negative Fobs − Fcalc maps were observed at the top and bottom of the oxygen atom, respectively, when the nitrogen atom was facing the catalytic center (Fig. 2A, b). The imidic acid form of Asn92, where the nitrogen atom faces the catalytic center, showed the weakest Fobs − Fcalc map in the x-ray analysis (Fig. 2A, d). In the neutron structure, strongly positive Fobs − Fcalc maps, which reflect deuteration/protonation of oxygen, were observed around the oxygen atom in the amide form of Asn92 (Fig. 2B, a and b). The hydrogen/deuterium (H/D) ratio was 0.3:0.7 under the crystallization conditions because of the difficulty of preparing fully deuterated 3MPD, and the H/D ratio of the imidic acid form of Asn92 is consistent with that value (Fig. 2B, d). The combined x-ray and neutron crystallography results indicate that the side chain of the asparagine residue takes the imidic acid form (HO–CX=NH), but not the typical amide form (O=CX–NH2), providing the first direct evidence that the imidic acid plays a key role in the hydrolysis.

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.