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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

SSADH substrate binding and the active site.A cartoon representation of the E. coli SSADH (monomer A: green) substrate (SSA) binding pocket superposed onto human SSADH C340A mutant with SSA bound (PDB ID: 2w8q [26]: red) and human SSADH containing the catalytic cysteine (PDB ID: 2w8o [26]: yellow). The key SSA binding residues from 2w8q have their interactions with SSA shown as a red dashed line. Superposition of catalytic residues are shown: the catalytic cysteine and the general base as sticks (labeled according the E. coli SSADH) and NADP+ (orange) from E. coli SSADH. It can be seen that the equivalent SSA binding residues from 2w8o and E. coli SSADH (R164, R283 and S445) are in a very similar location and orientation to those of 2w8q. The catalytic cysteine of 2w8o is oriented toward the NADP+ moiety while in E. coli C288 is oriented toward the substrate (SSA). Also two conformations of the general base (E254) can be seen in E. coli SSADH, with (a) being in the hydride conformation and (b) the hydrolysis conformation.
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pone-0009280-g005: SSADH substrate binding and the active site.A cartoon representation of the E. coli SSADH (monomer A: green) substrate (SSA) binding pocket superposed onto human SSADH C340A mutant with SSA bound (PDB ID: 2w8q [26]: red) and human SSADH containing the catalytic cysteine (PDB ID: 2w8o [26]: yellow). The key SSA binding residues from 2w8q have their interactions with SSA shown as a red dashed line. Superposition of catalytic residues are shown: the catalytic cysteine and the general base as sticks (labeled according the E. coli SSADH) and NADP+ (orange) from E. coli SSADH. It can be seen that the equivalent SSA binding residues from 2w8o and E. coli SSADH (R164, R283 and S445) are in a very similar location and orientation to those of 2w8q. The catalytic cysteine of 2w8o is oriented toward the NADP+ moiety while in E. coli C288 is oriented toward the substrate (SSA). Also two conformations of the general base (E254) can be seen in E. coli SSADH, with (a) being in the hydride conformation and (b) the hydrolysis conformation.

Mentions: E. coli SSADH was crystallised in the presence of both NADP+ and SSA. We observe no electron density for the substrate. However, superposition between human and E. coli SSADH does permit us to identify the substrate binding pocket (Figure 5).


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)

SSADH substrate binding and the active site.A cartoon representation of the E. coli SSADH (monomer A: green) substrate (SSA) binding pocket superposed onto human SSADH C340A mutant with SSA bound (PDB ID: 2w8q [26]: red) and human SSADH containing the catalytic cysteine (PDB ID: 2w8o [26]: yellow). The key SSA binding residues from 2w8q have their interactions with SSA shown as a red dashed line. Superposition of catalytic residues are shown: the catalytic cysteine and the general base as sticks (labeled according the E. coli SSADH) and NADP+ (orange) from E. coli SSADH. It can be seen that the equivalent SSA binding residues from 2w8o and E. coli SSADH (R164, R283 and S445) are in a very similar location and orientation to those of 2w8q. The catalytic cysteine of 2w8o is oriented toward the NADP+ moiety while in E. coli C288 is oriented toward the substrate (SSA). Also two conformations of the general base (E254) can be seen in E. coli SSADH, with (a) being in the hydride conformation and (b) the hydrolysis conformation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0009280-g005: SSADH substrate binding and the active site.A cartoon representation of the E. coli SSADH (monomer A: green) substrate (SSA) binding pocket superposed onto human SSADH C340A mutant with SSA bound (PDB ID: 2w8q [26]: red) and human SSADH containing the catalytic cysteine (PDB ID: 2w8o [26]: yellow). The key SSA binding residues from 2w8q have their interactions with SSA shown as a red dashed line. Superposition of catalytic residues are shown: the catalytic cysteine and the general base as sticks (labeled according the E. coli SSADH) and NADP+ (orange) from E. coli SSADH. It can be seen that the equivalent SSA binding residues from 2w8o and E. coli SSADH (R164, R283 and S445) are in a very similar location and orientation to those of 2w8q. The catalytic cysteine of 2w8o is oriented toward the NADP+ moiety while in E. coli C288 is oriented toward the substrate (SSA). Also two conformations of the general base (E254) can be seen in E. coli SSADH, with (a) being in the hydride conformation and (b) the hydrolysis conformation.
Mentions: E. coli SSADH was crystallised in the presence of both NADP+ and SSA. We observe no electron density for the substrate. However, superposition between human and E. coli SSADH does permit us to identify the substrate binding pocket (Figure 5).

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