Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Joseph S. Brunzelle; Douglas B. Jordan; Darrell R. McCaslin; Andrzej Olczak; Zdzislaw Wawrzak

doi:10.1016/j.abb.2008.03.007

Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Joseph S. Brunzelle, Douglas B. Jordan^*, Darrell R. McCaslin, Andrzej Olczak, Zdzislaw Wawrzak

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

50 Scopus citations

Abstract

The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 Å resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pK_a 7.2) serves as catalytic acid. The pH (6-10) profile for 1 / K_i^(BTP) is bell-shaped with pK_a's 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pK_a 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14^-E186^-. A sedimentation equilibrium experiment estimates a K_d ([dimer]²/[tetramer]) of 7 × 10^-9 M. Similar k_cat and k_cat/K_m values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

Original language	English (US)
Pages (from-to)	157-166
Number of pages	10
Journal	Archives of biochemistry and biophysics
Volume	474
Issue number	1
DOIs	https://doi.org/10.1016/j.abb.2008.03.007
State	Published - Jun 1 2008

Funding

We thank Jay D. Braker for excellent technical assistance. Sedimentation equilibrium data was collected in Biophysics Instrumentation Facility at the University of Wisconsin (Madison), which was established with support from the University of Wisconsin, and Grants BIR-9512577 (NSF) and S10 RR13790 (NIH). This work was supported by USDA funding of CRIS 3620-41000-118-00D. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.

Keywords

GH43
Glycoside hydrolase
Protein crystallography
Sedimentation equilibrium
Structure-function
α-l-Arabinofuranosidase

ASJC Scopus subject areas

Molecular Biology
Biophysics
Biochemistry

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10.1016/j.abb.2008.03.007

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title = "Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane",

abstract = "The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 {\AA} resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pKa 7.2) serves as catalytic acid. The pH (6-10) profile for 1 / Ki(BTP) is bell-shaped with pKa's 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pKa 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14-E186-. A sedimentation equilibrium experiment estimates a Kd ([dimer]2/[tetramer]) of 7 × 10-9 M. Similar kcat and kcat/Km values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.",

keywords = "GH43, Glycoside hydrolase, Protein crystallography, Sedimentation equilibrium, Structure-function, α-l-Arabinofuranosidase",

author = "Brunzelle, {Joseph S.} and Jordan, {Douglas B.} and McCaslin, {Darrell R.} and Andrzej Olczak and Zdzislaw Wawrzak",

note = "Funding Information: We thank Jay D. Braker for excellent technical assistance. Sedimentation equilibrium data was collected in Biophysics Instrumentation Facility at the University of Wisconsin (Madison), which was established with support from the University of Wisconsin, and Grants BIR-9512577 (NSF) and S10 RR13790 (NIH). This work was supported by USDA funding of CRIS 3620-41000-118-00D. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. ",

year = "2008",

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AU - Jordan, Douglas B.

AU - McCaslin, Darrell R.

AU - Olczak, Andrzej

AU - Wawrzak, Zdzislaw

N1 - Funding Information: We thank Jay D. Braker for excellent technical assistance. Sedimentation equilibrium data was collected in Biophysics Instrumentation Facility at the University of Wisconsin (Madison), which was established with support from the University of Wisconsin, and Grants BIR-9512577 (NSF) and S10 RR13790 (NIH). This work was supported by USDA funding of CRIS 3620-41000-118-00D. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.

PY - 2008/6/1

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N2 - The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 Å resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pKa 7.2) serves as catalytic acid. The pH (6-10) profile for 1 / Ki(BTP) is bell-shaped with pKa's 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pKa 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14-E186-. A sedimentation equilibrium experiment estimates a Kd ([dimer]2/[tetramer]) of 7 × 10-9 M. Similar kcat and kcat/Km values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

AB - The three-dimensional structure of the catalytically efficient β-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3-bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 Å resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. E186 (pKa 7.2) serves as catalytic acid. The pH (6-10) profile for 1 / Ki(BTP) is bell-shaped with pKa's 6.8 and 7.8 on the acidic limb assigned to E186 and inhibitor groups and 9.9 on the basic limb assigned to inhibitor. Mutation K99A eliminates pKa 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14-E186-. A sedimentation equilibrium experiment estimates a Kd ([dimer]2/[tetramer]) of 7 × 10-9 M. Similar kcat and kcat/Km values were determined when the tetramer/dimer ratio changes from 0.0028 to 26 suggesting that dimers and tetramers are equally active forms.

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KW - Sedimentation equilibrium

KW - Structure-function

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Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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Structure of the two subsite D-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Cite this

Structure of the two-subsite β-d-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Datasets

Structure of the two subsite D-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino]propane

Cite this