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The X-ray crystal structure of Escherichia coli succinic semialdehyde dehydrogenase; structural insights into NADP+/enzyme interactions.

Langendorf CG, Key TL, Fenalti G, Kan WT, Buckle AM, Caradoc-Davies T, Tuck KL, Law RH, Whisstock JC - PLoS ONE (2010)

Bottom Line: In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+.Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia.

ABSTRACT

Background: In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and gamma-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.

Methodology/principal findings: Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.

Conclusions/significance: Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.

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Related in: MedlinePlus

Crystal structure of E. coli SSADH.a) Two cartoon representations of E. coli SSADH monomer (rotated by 180°) with NADP+ bound (orange) comprises of the catalytic domain (blue and light blue) with catalytic loop (red), the cofactor binding domain (green and yellow, where yellow illustrates the Rossmann fold) and the oligomerisation domain (magenta); b) A cartoon representation of the E. coli SSADH dimer with NADP+ bound (orange), it can be seen that the 3-stranded oligomerisation domain β- sheet (dark green) of the green monomer is extending the 7-stranded catalytic domain β- sheet (dark blue) of the blue monomer to form a 10-stranded β- sheet. c) Two surface representation models of the SSADH tetramer (rotated by 180°) showing the dimer of dimer formation between the blue and red monomer and the green and light blue monomers. NADP+ (orange) can be seen on the same face of the dimer, the substrate binding pocket has also been labelled.
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pone-0009280-g002: Crystal structure of E. coli SSADH.a) Two cartoon representations of E. coli SSADH monomer (rotated by 180°) with NADP+ bound (orange) comprises of the catalytic domain (blue and light blue) with catalytic loop (red), the cofactor binding domain (green and yellow, where yellow illustrates the Rossmann fold) and the oligomerisation domain (magenta); b) A cartoon representation of the E. coli SSADH dimer with NADP+ bound (orange), it can be seen that the 3-stranded oligomerisation domain β- sheet (dark green) of the green monomer is extending the 7-stranded catalytic domain β- sheet (dark blue) of the blue monomer to form a 10-stranded β- sheet. c) Two surface representation models of the SSADH tetramer (rotated by 180°) showing the dimer of dimer formation between the blue and red monomer and the green and light blue monomers. NADP+ (orange) can be seen on the same face of the dimer, the substrate binding pocket has also been labelled.

Mentions: The structure of E. coli SSADH reveals four monomers (A–D, 481 amino acid per monomer) in the asymmetric unit (Figure 2), forming, like other members of the aldehyde dehydrogenase (ALDH) family, a biologically relevant homotetramer [28], [29], [30], [31], [32] with the 4 monomers related by a non-crystallographic 222 symmetry. The four monomers can be superposed with root-mean-square deviation (r.m.s.d: over all Cα's) of 0.193 to 0.377 Å. Monomers AB and CD form obligate dimers, which then assemble into a functional tetramer [32]. For the initial description of the structure, we refer primarily to monomer A.


The X-ray crystal structure of Escherichia coli succinic semialdehyde dehydrogenase; structural insights into NADP+/enzyme interactions.

Langendorf CG, Key TL, Fenalti G, Kan WT, Buckle AM, Caradoc-Davies T, Tuck KL, Law RH, Whisstock JC - PLoS ONE (2010)

Crystal structure of E. coli SSADH.a) Two cartoon representations of E. coli SSADH monomer (rotated by 180°) with NADP+ bound (orange) comprises of the catalytic domain (blue and light blue) with catalytic loop (red), the cofactor binding domain (green and yellow, where yellow illustrates the Rossmann fold) and the oligomerisation domain (magenta); b) A cartoon representation of the E. coli SSADH dimer with NADP+ bound (orange), it can be seen that the 3-stranded oligomerisation domain β- sheet (dark green) of the green monomer is extending the 7-stranded catalytic domain β- sheet (dark blue) of the blue monomer to form a 10-stranded β- sheet. c) Two surface representation models of the SSADH tetramer (rotated by 180°) showing the dimer of dimer formation between the blue and red monomer and the green and light blue monomers. NADP+ (orange) can be seen on the same face of the dimer, the substrate binding pocket has also been labelled.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2823781&req=5

pone-0009280-g002: Crystal structure of E. coli SSADH.a) Two cartoon representations of E. coli SSADH monomer (rotated by 180°) with NADP+ bound (orange) comprises of the catalytic domain (blue and light blue) with catalytic loop (red), the cofactor binding domain (green and yellow, where yellow illustrates the Rossmann fold) and the oligomerisation domain (magenta); b) A cartoon representation of the E. coli SSADH dimer with NADP+ bound (orange), it can be seen that the 3-stranded oligomerisation domain β- sheet (dark green) of the green monomer is extending the 7-stranded catalytic domain β- sheet (dark blue) of the blue monomer to form a 10-stranded β- sheet. c) Two surface representation models of the SSADH tetramer (rotated by 180°) showing the dimer of dimer formation between the blue and red monomer and the green and light blue monomers. NADP+ (orange) can be seen on the same face of the dimer, the substrate binding pocket has also been labelled.
Mentions: The structure of E. coli SSADH reveals four monomers (A–D, 481 amino acid per monomer) in the asymmetric unit (Figure 2), forming, like other members of the aldehyde dehydrogenase (ALDH) family, a biologically relevant homotetramer [28], [29], [30], [31], [32] with the 4 monomers related by a non-crystallographic 222 symmetry. The four monomers can be superposed with root-mean-square deviation (r.m.s.d: over all Cα's) of 0.193 to 0.377 Å. Monomers AB and CD form obligate dimers, which then assemble into a functional tetramer [32]. For the initial description of the structure, we refer primarily to monomer A.

Bottom Line: In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+.Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria, Australia.

ABSTRACT

Background: In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and gamma-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.

Methodology/principal findings: Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.

Conclusions/significance: Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.

Show MeSH
Related in: MedlinePlus