Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparisons between human (PDB ID: 2w8r)[26] and E.coli (monomer A) SSADH cofactor binding and SSA binding pockets, visualised using electrostatic surface representations (red represents negatively charged surfaces and blue represents positively charged surfaces).A–B) both human (A: NAD+ in yellow) and E. coli (B: NADP+ in orange) SSADH have a two pocket NAD(P)+ binding site per molecule, the first (mostly blue), positively charged and close to the surface, accommodates the adenosine moiety (and the 2'phosphate in E. coli SSADH). The second binding pocket, deep in the active site, houses the nicotinamide ribose moiety (absent in human SSADH). The smaller human SSADH cofactor binding pocket has a large positive protrusion, which closes the bottom of the pocket, while the larger E. coli SSADH cofactor binding pocket can clearly accommodate the 2'phosphate of the NADP+. C–D) shows the positively charged SSA binding pocket of both human (C) and E. coli (D) SSADH highlighted by a white dashed line. The human SSA binding pocket is larger than the E. coli SSA binding pocket.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2823781&req=5

pone-0009280-g004: Comparisons between human (PDB ID: 2w8r)[26] and E.coli (monomer A) SSADH cofactor binding and SSA binding pockets, visualised using electrostatic surface representations (red represents negatively charged surfaces and blue represents positively charged surfaces).A–B) both human (A: NAD+ in yellow) and E. coli (B: NADP+ in orange) SSADH have a two pocket NAD(P)+ binding site per molecule, the first (mostly blue), positively charged and close to the surface, accommodates the adenosine moiety (and the 2'phosphate in E. coli SSADH). The second binding pocket, deep in the active site, houses the nicotinamide ribose moiety (absent in human SSADH). The smaller human SSADH cofactor binding pocket has a large positive protrusion, which closes the bottom of the pocket, while the larger E. coli SSADH cofactor binding pocket can clearly accommodate the 2'phosphate of the NADP+. C–D) shows the positively charged SSA binding pocket of both human (C) and E. coli (D) SSADH highlighted by a white dashed line. The human SSA binding pocket is larger than the E. coli SSA binding pocket.

Mentions: In each SSADH monomer, the catalytic residues are located at the centre of the molecule with two funnel-like openings on the surface of either side of the molecule. The larger opening functions to allow entry of the cofactor NADP+ (Figure 4A, B). On the opposite side of the monomer, the smaller opening is located, and this cavity is utilized for substrate entry and product exit (Figure 4C, D, 2C) as for other ALDHs.


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)

Comparisons between human (PDB ID: 2w8r)[26] and E.coli (monomer A) SSADH cofactor binding and SSA binding pockets, visualised using electrostatic surface representations (red represents negatively charged surfaces and blue represents positively charged surfaces).A–B) both human (A: NAD+ in yellow) and E. coli (B: NADP+ in orange) SSADH have a two pocket NAD(P)+ binding site per molecule, the first (mostly blue), positively charged and close to the surface, accommodates the adenosine moiety (and the 2'phosphate in E. coli SSADH). The second binding pocket, deep in the active site, houses the nicotinamide ribose moiety (absent in human SSADH). The smaller human SSADH cofactor binding pocket has a large positive protrusion, which closes the bottom of the pocket, while the larger E. coli SSADH cofactor binding pocket can clearly accommodate the 2'phosphate of the NADP+. C–D) shows the positively charged SSA binding pocket of both human (C) and E. coli (D) SSADH highlighted by a white dashed line. The human SSA binding pocket is larger than the E. coli SSA binding pocket.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009280-g004: Comparisons between human (PDB ID: 2w8r)[26] and E.coli (monomer A) SSADH cofactor binding and SSA binding pockets, visualised using electrostatic surface representations (red represents negatively charged surfaces and blue represents positively charged surfaces).A–B) both human (A: NAD+ in yellow) and E. coli (B: NADP+ in orange) SSADH have a two pocket NAD(P)+ binding site per molecule, the first (mostly blue), positively charged and close to the surface, accommodates the adenosine moiety (and the 2'phosphate in E. coli SSADH). The second binding pocket, deep in the active site, houses the nicotinamide ribose moiety (absent in human SSADH). The smaller human SSADH cofactor binding pocket has a large positive protrusion, which closes the bottom of the pocket, while the larger E. coli SSADH cofactor binding pocket can clearly accommodate the 2'phosphate of the NADP+. C–D) shows the positively charged SSA binding pocket of both human (C) and E. coli (D) SSADH highlighted by a white dashed line. The human SSA binding pocket is larger than the E. coli SSA binding pocket.
Mentions: In each SSADH monomer, the catalytic residues are located at the centre of the molecule with two funnel-like openings on the surface of either side of the molecule. The larger opening functions to allow entry of the cofactor NADP+ (Figure 4A, B). On the opposite side of the monomer, the smaller opening is located, and this cavity is utilized for substrate entry and product exit (Figure 4C, D, 2C) as for other ALDHs.

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