Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism

Joseph J Barycki, Laurie K. O'Brien, Judy M. Bratt, Rongguang Zhang, Ruslan Sanishvili, Arnold W. Strauss, Leonard J. Banaszak

Research output: Contribution to journalArticle

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Abstract

Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the β-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other α/β dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central β-sheet. The second, or C-terminal, domain is primarily α-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.

Original languageEnglish (US)
Pages (from-to)5786-5798
Number of pages13
JournalBiochemistry
Volume38
Issue number18
DOIs
StatePublished - May 4 1999

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3-Hydroxyacyl-CoA Dehydrogenase
NAD
Crystal structure
Coenzyme A
Enzymes
Helix-Turn-Helix Motifs
Selenomethionine
Oxidation
Molecular modeling
Dimerization
Metabolic Diseases
Coulomb interactions
Static Electricity
Hydroxyl Radical
Escherichia coli
Protons
Diffraction
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism. / Barycki, Joseph J; O'Brien, Laurie K.; Bratt, Judy M.; Zhang, Rongguang; Sanishvili, Ruslan; Strauss, Arnold W.; Banaszak, Leonard J.

In: Biochemistry, Vol. 38, No. 18, 04.05.1999, p. 5786-5798.

Research output: Contribution to journalArticle

Barycki, Joseph J ; O'Brien, Laurie K. ; Bratt, Judy M. ; Zhang, Rongguang ; Sanishvili, Ruslan ; Strauss, Arnold W. ; Banaszak, Leonard J. / Biochemical characterization and crystal structure determination of human heart short chain L-3-hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism. In: Biochemistry. 1999 ; Vol. 38, No. 18. pp. 5786-5798.
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abstract = "Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the β-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other α/β dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central β-sheet. The second, or C-terminal, domain is primarily α-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.",
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AU - Sanishvili, Ruslan

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AU - Banaszak, Leonard J.

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N2 - Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the β-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other α/β dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central β-sheet. The second, or C-terminal, domain is primarily α-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.

AB - Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD+ to NADH, as part of the β-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other α/β dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central β-sheet. The second, or C-terminal, domain is primarily α-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD+ complex suggest that His 158 serves as a general base, abstracting a proton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.

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