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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.


Related in: MedlinePlus

Proton relay stabilizing the imidic acid state of Asn92.(A) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn92 and the amide of Cys96. The 2Fobs − Fcalc (blue) and Fobs − Fcalc (red and green) maps of the x-ray analysis are shown at the 1.0σ and 3.0σ levels, respectively, and 2Fobs − Fcalc (purple) and Fobs − Fcalc (red and green) maps of the neutron analysis are shown at the 1.0σ and 2.0σ levels, respectively. (B) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn105 and amide of Asn105, His107, and Met109. The 2Fobs − Fcalc map of the x-ray analysis is shown at the 1.0σ level, and the Fobs − Fcalc map of the neutron analysis is shown at the 2.0σ level. (C) Proposed mechanism of formation of the imidic acid form of Asn92.
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Figure 3: Proton relay stabilizing the imidic acid state of Asn92.(A) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn92 and the amide of Cys96. The 2Fobs − Fcalc (blue) and Fobs − Fcalc (red and green) maps of the x-ray analysis are shown at the 1.0σ and 3.0σ levels, respectively, and 2Fobs − Fcalc (purple) and Fobs − Fcalc (red and green) maps of the neutron analysis are shown at the 1.0σ and 2.0σ levels, respectively. (B) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn105 and amide of Asn105, His107, and Met109. The 2Fobs − Fcalc map of the x-ray analysis is shown at the 1.0σ level, and the Fobs − Fcalc map of the neutron analysis is shown at the 2.0σ level. (C) Proposed mechanism of formation of the imidic acid form of Asn92.

Mentions: The pKa value for protonation of carbonyl oxygen of the imidic acid form was estimated as around 0 (10), and that of protonation/deprotonation of nitrogen was estimated as 4.5 to 7.5 (11, 12). Thus, the nitrogen atom of the imidic acid form of asparagine can act as a base in PcCel45A, although it is not yet clear how the oxygen atom of asparagine is protonated. The x-ray and neutron H/D omit maps around the imidic acid of Asn92 are shown in Fig. 3A. The positive Fobs − Fcalc map in the neutron analysis connects the nitrogen atoms of neighboring amides of Cys96 and oxygen atoms of side and main chains of Asn92, although nothing was observed in the x-ray structure. Considering that x-rays are scattered by electrons, whereas neutrons are scattered by the nucleus, protons/deuterons only show up in the neutron diffraction analysis. For example, the positions of protons in zeolite were determined by neutron powder diffraction analysis (13), and the proton dynamics in N-methylacetamide was studied by inelastic neutron scattering (14). Intramolecular proton transfer at hydrogen bonds between carbonyl and amide of small molecules often occurs if the positions of nitrogen and oxygen are suitable (15). In addition, the possibility of proton transfer between protonated carbonyl of the main chain and carbonyl of the side chain of Asn92 was indicated by a theoretical study of analogs of malonaldehyde (16). These results all support the idea that proton/deuteron transfer from the amide of Cys96 to the carbonyl of the side chain of Asn92 can occur, and this would be consistent with stabilization of the imidic acid form of Asn92 by multiple tautomerizations at the surface of the enzyme, that is, the side-chain carbonyl group of Asn105 is highly protonated/deuterated by a “proton sink” consisting of amides of His107, Met109, and the main chain of Asn105 (Fig. 3B), and thus, the side-chain amide group of Asn105 can transfer a proton/deuteron to carbonyl of Phe95 via the imidic acid form, such as Asn92, and the amide of Cys96 passes the proton/deuteron to carbonyl of Asn92 (Fig. 3C). The proton network originating from the proton sink was not seen at all in subatomic x-ray structures PcCel45A (~0.64 Å), emphasizing the importance of neutron crystallography for visualizing the localization of protons.


"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)

Proton relay stabilizing the imidic acid state of Asn92.(A) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn92 and the amide of Cys96. The 2Fobs − Fcalc (blue) and Fobs − Fcalc (red and green) maps of the x-ray analysis are shown at the 1.0σ and 3.0σ levels, respectively, and 2Fobs − Fcalc (purple) and Fobs − Fcalc (red and green) maps of the neutron analysis are shown at the 1.0σ and 2.0σ levels, respectively. (B) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn105 and amide of Asn105, His107, and Met109. The 2Fobs − Fcalc map of the x-ray analysis is shown at the 1.0σ level, and the Fobs − Fcalc map of the neutron analysis is shown at the 2.0σ level. (C) Proposed mechanism of formation of the imidic acid form of Asn92.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Proton relay stabilizing the imidic acid state of Asn92.(A) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn92 and the amide of Cys96. The 2Fobs − Fcalc (blue) and Fobs − Fcalc (red and green) maps of the x-ray analysis are shown at the 1.0σ and 3.0σ levels, respectively, and 2Fobs − Fcalc (purple) and Fobs − Fcalc (red and green) maps of the neutron analysis are shown at the 1.0σ and 2.0σ levels, respectively. (B) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn105 and amide of Asn105, His107, and Met109. The 2Fobs − Fcalc map of the x-ray analysis is shown at the 1.0σ level, and the Fobs − Fcalc map of the neutron analysis is shown at the 2.0σ level. (C) Proposed mechanism of formation of the imidic acid form of Asn92.
Mentions: The pKa value for protonation of carbonyl oxygen of the imidic acid form was estimated as around 0 (10), and that of protonation/deprotonation of nitrogen was estimated as 4.5 to 7.5 (11, 12). Thus, the nitrogen atom of the imidic acid form of asparagine can act as a base in PcCel45A, although it is not yet clear how the oxygen atom of asparagine is protonated. The x-ray and neutron H/D omit maps around the imidic acid of Asn92 are shown in Fig. 3A. The positive Fobs − Fcalc map in the neutron analysis connects the nitrogen atoms of neighboring amides of Cys96 and oxygen atoms of side and main chains of Asn92, although nothing was observed in the x-ray structure. Considering that x-rays are scattered by electrons, whereas neutrons are scattered by the nucleus, protons/deuterons only show up in the neutron diffraction analysis. For example, the positions of protons in zeolite were determined by neutron powder diffraction analysis (13), and the proton dynamics in N-methylacetamide was studied by inelastic neutron scattering (14). Intramolecular proton transfer at hydrogen bonds between carbonyl and amide of small molecules often occurs if the positions of nitrogen and oxygen are suitable (15). In addition, the possibility of proton transfer between protonated carbonyl of the main chain and carbonyl of the side chain of Asn92 was indicated by a theoretical study of analogs of malonaldehyde (16). These results all support the idea that proton/deuteron transfer from the amide of Cys96 to the carbonyl of the side chain of Asn92 can occur, and this would be consistent with stabilization of the imidic acid form of Asn92 by multiple tautomerizations at the surface of the enzyme, that is, the side-chain carbonyl group of Asn105 is highly protonated/deuterated by a “proton sink” consisting of amides of His107, Met109, and the main chain of Asn105 (Fig. 3B), and thus, the side-chain amide group of Asn105 can transfer a proton/deuteron to carbonyl of Phe95 via the imidic acid form, such as Asn92, and the amide of Cys96 passes the proton/deuteron to carbonyl of Asn92 (Fig. 3C). The proton network originating from the proton sink was not seen at all in subatomic x-ray structures PcCel45A (~0.64 Å), emphasizing the importance of neutron crystallography for visualizing the localization of protons.

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.


Related in: MedlinePlus