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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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A single molecule superposition of E. coli SSADH and human SSADH.A) Cα trace of monomer A of E. coli SSADH (green) superposed with the human SSADH molecule (PDB ID: 2w8r [26]: yellow: r.m.s.d. = 0.712 over 473 residues), with the NADP+ moiety (orange) from E. coli SSADH. Two structurally variable regions have been highlighted with dashed lines and labelled B–C. Figures B–C show Cα traces of all four E. coli SSADH monomers A–D (green) and 5 human SSADH monomers (open loop, PDB ID: 2w8o, 2w8p, 2w8q, 2w8r yellow; closed loop, PDB ID: 2w8n magenta)[26] superposed onto each other, only one NADP+ molecule (orange) from monomer A of E. coli SSADH is shown. B) Shows the region surrounding the 3 amino acid insertion (261RKN263) in human SSADH, which clashes with the 2'phosphate of NADP+. C) The loop motif connecting s2D and s3D in the catalytic domain, residues A379–G388 in E. coli SSADH and M432–G441 in human SSADH (r.m.s.d. of 3.4 Å over 10 residues). The E. coli SSADH loop is conserved throughout the ALDH family and is stabilised by 7 hydrogen bonds. The novel loop in human SSADH is stabilised by only 3 hydrogen bonds, furthermore this same loop in the reduced wild type human SSADH (PDB ID: 2w8o)[26] is highly flexible and could not be determined using X-ray crystallography.
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pone-0009280-g003: A single molecule superposition of E. coli SSADH and human SSADH.A) Cα trace of monomer A of E. coli SSADH (green) superposed with the human SSADH molecule (PDB ID: 2w8r [26]: yellow: r.m.s.d. = 0.712 over 473 residues), with the NADP+ moiety (orange) from E. coli SSADH. Two structurally variable regions have been highlighted with dashed lines and labelled B–C. Figures B–C show Cα traces of all four E. coli SSADH monomers A–D (green) and 5 human SSADH monomers (open loop, PDB ID: 2w8o, 2w8p, 2w8q, 2w8r yellow; closed loop, PDB ID: 2w8n magenta)[26] superposed onto each other, only one NADP+ molecule (orange) from monomer A of E. coli SSADH is shown. B) Shows the region surrounding the 3 amino acid insertion (261RKN263) in human SSADH, which clashes with the 2'phosphate of NADP+. C) The loop motif connecting s2D and s3D in the catalytic domain, residues A379–G388 in E. coli SSADH and M432–G441 in human SSADH (r.m.s.d. of 3.4 Å over 10 residues). The E. coli SSADH loop is conserved throughout the ALDH family and is stabilised by 7 hydrogen bonds. The novel loop in human SSADH is stabilised by only 3 hydrogen bonds, furthermore this same loop in the reduced wild type human SSADH (PDB ID: 2w8o)[26] is highly flexible and could not be determined using X-ray crystallography.

Mentions: The structure of human SSADH in both the active (open, reduced; PDB ID: 2w8o) and inactive (closed, oxidised; PDB ID: 2w8n) state has recently been determined [26]. E. coli SSADH was purified and crystallised in the presence of the reducing agent β-mercaptoethanol, and accordingly, the structure we report most closely resembles the active form of human SSADH (2w8o) and superposes with a root-mean-square deviation of 0.79 Å over 472 Cα (2w8o and Monomer A, Figure 3A and Figure S2). The structure of the catalytic loop in E. coli and human SSADH is essentially identical, furthermore, the two cysteine residues involved in the redox switch in human SSADH are conserved in E. coli (Figure S2). These data suggest that E. coli SSADH may also be regulated via the redox status of the surrounding milieu. Significantly, our results show that E. coli gabD gene product is inactive in the presence of H2O2 and can be reactivated upon addition of DTT (Figure S3). Interestingly, the other E. coli SSADH gene, sad, does not contain the dual conserved cysteine residues in the catalytic loop and therefore it may not be regulated via the same redox mechanism.


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)

A single molecule superposition of E. coli SSADH and human SSADH.A) Cα trace of monomer A of E. coli SSADH (green) superposed with the human SSADH molecule (PDB ID: 2w8r [26]: yellow: r.m.s.d. = 0.712 over 473 residues), with the NADP+ moiety (orange) from E. coli SSADH. Two structurally variable regions have been highlighted with dashed lines and labelled B–C. Figures B–C show Cα traces of all four E. coli SSADH monomers A–D (green) and 5 human SSADH monomers (open loop, PDB ID: 2w8o, 2w8p, 2w8q, 2w8r yellow; closed loop, PDB ID: 2w8n magenta)[26] superposed onto each other, only one NADP+ molecule (orange) from monomer A of E. coli SSADH is shown. B) Shows the region surrounding the 3 amino acid insertion (261RKN263) in human SSADH, which clashes with the 2'phosphate of NADP+. C) The loop motif connecting s2D and s3D in the catalytic domain, residues A379–G388 in E. coli SSADH and M432–G441 in human SSADH (r.m.s.d. of 3.4 Å over 10 residues). The E. coli SSADH loop is conserved throughout the ALDH family and is stabilised by 7 hydrogen bonds. The novel loop in human SSADH is stabilised by only 3 hydrogen bonds, furthermore this same loop in the reduced wild type human SSADH (PDB ID: 2w8o)[26] is highly flexible and could not be determined using X-ray crystallography.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2823781&req=5

pone-0009280-g003: A single molecule superposition of E. coli SSADH and human SSADH.A) Cα trace of monomer A of E. coli SSADH (green) superposed with the human SSADH molecule (PDB ID: 2w8r [26]: yellow: r.m.s.d. = 0.712 over 473 residues), with the NADP+ moiety (orange) from E. coli SSADH. Two structurally variable regions have been highlighted with dashed lines and labelled B–C. Figures B–C show Cα traces of all four E. coli SSADH monomers A–D (green) and 5 human SSADH monomers (open loop, PDB ID: 2w8o, 2w8p, 2w8q, 2w8r yellow; closed loop, PDB ID: 2w8n magenta)[26] superposed onto each other, only one NADP+ molecule (orange) from monomer A of E. coli SSADH is shown. B) Shows the region surrounding the 3 amino acid insertion (261RKN263) in human SSADH, which clashes with the 2'phosphate of NADP+. C) The loop motif connecting s2D and s3D in the catalytic domain, residues A379–G388 in E. coli SSADH and M432–G441 in human SSADH (r.m.s.d. of 3.4 Å over 10 residues). The E. coli SSADH loop is conserved throughout the ALDH family and is stabilised by 7 hydrogen bonds. The novel loop in human SSADH is stabilised by only 3 hydrogen bonds, furthermore this same loop in the reduced wild type human SSADH (PDB ID: 2w8o)[26] is highly flexible and could not be determined using X-ray crystallography.
Mentions: The structure of human SSADH in both the active (open, reduced; PDB ID: 2w8o) and inactive (closed, oxidised; PDB ID: 2w8n) state has recently been determined [26]. E. coli SSADH was purified and crystallised in the presence of the reducing agent β-mercaptoethanol, and accordingly, the structure we report most closely resembles the active form of human SSADH (2w8o) and superposes with a root-mean-square deviation of 0.79 Å over 472 Cα (2w8o and Monomer A, Figure 3A and Figure S2). The structure of the catalytic loop in E. coli and human SSADH is essentially identical, furthermore, the two cysteine residues involved in the redox switch in human SSADH are conserved in E. coli (Figure S2). These data suggest that E. coli SSADH may also be regulated via the redox status of the surrounding milieu. Significantly, our results show that E. coli gabD gene product is inactive in the presence of H2O2 and can be reactivated upon addition of DTT (Figure S3). Interestingly, the other E. coli SSADH gene, sad, does not contain the dual conserved cysteine residues in the catalytic loop and therefore it may not be regulated via the same redox mechanism.

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