Mechanism of the Reaction Catalyzed by Mandelate Racemase. 2. Crystal Structure of Mandelate Racemase at 2.5-Å Resolution: Identification of the Active Site and Possible Catalytic Residues

David J. Neidhart, P. Lynne Howell, Gregory A. Petsko, Vincent M. Powers, Rongshi Li, George L. Kenyon, John A. Gerlt

Research output: Contribution to journalArticle

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Abstract

The crystal structure of mandelate racemase (MR) has been solved at 3.0-Å resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-Å resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an α + β topology consisting of a three-stranded antiparallel β-sheet followed by an antiparallel four α-helix bundle. The central domain is a singly wound parallel α/β-barrel composed of eight central strands of β-sheet and seven α-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel β-sheet and two short α-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eighth α-helix in all other known parallel α/β-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the β-strands in the α/β-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central β-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-Å resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts. The results of these structural studies support the conclusions reached from the chemical and kinetic experiments described in the preceding paper regarding the number and chemical properties of the bases in the active site of MR [Powers, V. M., Koo, C., Kenyon, G. K., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue)]. This is the first three-dimensional structure of a racemase to be determined.

Original languageEnglish (US)
Pages (from-to)9264-9273
Number of pages10
JournalBiochemistry
Volume30
Issue number38
DOIs
StatePublished - Sep 1 1991

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mandelate racemase
Catalytic Domain
Crystal structure
R388
Substrates
Racemases and Epimerases
Biochemistry
Fourier analysis
Fourier Analysis
Enzymes
Molecular Dynamics Simulation
Least-Squares Analysis
Catalysis
Catalyst supports
X-Ray Diffraction
Chemical properties
Metal ions
Molecular dynamics
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry

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Mechanism of the Reaction Catalyzed by Mandelate Racemase. 2. Crystal Structure of Mandelate Racemase at 2.5-Å Resolution : Identification of the Active Site and Possible Catalytic Residues. / Neidhart, David J.; Howell, P. Lynne; Petsko, Gregory A.; Powers, Vincent M.; Li, Rongshi; Kenyon, George L.; Gerlt, John A.

In: Biochemistry, Vol. 30, No. 38, 01.09.1991, p. 9264-9273.

Research output: Contribution to journalArticle

Neidhart, David J. ; Howell, P. Lynne ; Petsko, Gregory A. ; Powers, Vincent M. ; Li, Rongshi ; Kenyon, George L. ; Gerlt, John A. / Mechanism of the Reaction Catalyzed by Mandelate Racemase. 2. Crystal Structure of Mandelate Racemase at 2.5-Å Resolution : Identification of the Active Site and Possible Catalytic Residues. In: Biochemistry. 1991 ; Vol. 30, No. 38. pp. 9264-9273.
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abstract = "The crystal structure of mandelate racemase (MR) has been solved at 3.0-{\AA} resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-{\AA} resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3{\%}. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an α + β topology consisting of a three-stranded antiparallel β-sheet followed by an antiparallel four α-helix bundle. The central domain is a singly wound parallel α/β-barrel composed of eight central strands of β-sheet and seven α-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel β-sheet and two short α-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eighth α-helix in all other known parallel α/β-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the β-strands in the α/β-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central β-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-{\AA} resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts. The results of these structural studies support the conclusions reached from the chemical and kinetic experiments described in the preceding paper regarding the number and chemical properties of the bases in the active site of MR [Powers, V. M., Koo, C., Kenyon, G. K., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue)]. This is the first three-dimensional structure of a racemase to be determined.",
author = "Neidhart, {David J.} and Howell, {P. Lynne} and Petsko, {Gregory A.} and Powers, {Vincent M.} and Rongshi Li and Kenyon, {George L.} and Gerlt, {John A.}",
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T1 - Mechanism of the Reaction Catalyzed by Mandelate Racemase. 2. Crystal Structure of Mandelate Racemase at 2.5-Å Resolution

T2 - Identification of the Active Site and Possible Catalytic Residues

AU - Neidhart, David J.

AU - Howell, P. Lynne

AU - Petsko, Gregory A.

AU - Powers, Vincent M.

AU - Li, Rongshi

AU - Kenyon, George L.

AU - Gerlt, John A.

PY - 1991/9/1

Y1 - 1991/9/1

N2 - The crystal structure of mandelate racemase (MR) has been solved at 3.0-Å resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-Å resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an α + β topology consisting of a three-stranded antiparallel β-sheet followed by an antiparallel four α-helix bundle. The central domain is a singly wound parallel α/β-barrel composed of eight central strands of β-sheet and seven α-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel β-sheet and two short α-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eighth α-helix in all other known parallel α/β-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the β-strands in the α/β-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central β-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-Å resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts. The results of these structural studies support the conclusions reached from the chemical and kinetic experiments described in the preceding paper regarding the number and chemical properties of the bases in the active site of MR [Powers, V. M., Koo, C., Kenyon, G. K., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue)]. This is the first three-dimensional structure of a racemase to be determined.

AB - The crystal structure of mandelate racemase (MR) has been solved at 3.0-Å resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-Å resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an α + β topology consisting of a three-stranded antiparallel β-sheet followed by an antiparallel four α-helix bundle. The central domain is a singly wound parallel α/β-barrel composed of eight central strands of β-sheet and seven α-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel β-sheet and two short α-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eighth α-helix in all other known parallel α/β-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the β-strands in the α/β-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central β-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-Å resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts. The results of these structural studies support the conclusions reached from the chemical and kinetic experiments described in the preceding paper regarding the number and chemical properties of the bases in the active site of MR [Powers, V. M., Koo, C., Kenyon, G. K., Gerlt, J. A., & Kozarich, J. W. (1991) Biochemistry (first paper of three in this issue)]. This is the first three-dimensional structure of a racemase to be determined.

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