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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

NADP+ binding of E. coli SSADH.Stereo view of the active site showing the NADP+ moiety (yellow), SSADH residues (green) involved in binding NADP+, water molecules can be seen as red spheres and all bonds are depicted with a black dashed line. The 2F0–Fc omit electron density of the NADP+ moiety contoured at 1σ is also shown (light blue mesh). Interactions of monomer A and NADP+ can be seen, specifically both AN1 and AN6 of the adenine moiety (labelled adn) interacts with Q239(Oε1), Q243(Oε1) and N217(Oδ1) via water molecules. Adjacent to the adenine moiety, both AO2 and AO3 of the ribose (labelled rb2) hydrogen bonds with T153(O) and K179(NZ, O) via a single water molecule. 3AOP of the 2'-phosphate interacts with K179(NZ). AO2 of the pyrophosphate interacts with S233(N, OG) and the NO1 hydrogen bonds directly with W155(Nε1). Both NO2 and NO3 of the adjacent ribose moiety (labelled rb1) hydrogen bonds with K338(NZ), while NO2 also interacts with E385(Oε1). NN7 of the nicotinamide (labelled nt) moiety interacts with N156(Nδ2) and the catalytic C288(N) via the single water molecule. While NO7 interacts directly with G232(O) and L255(O), as well as with L255(N) and E254(Oε1) via the same water molecule. Up to 13 SSADH residues make 24 van der Waals or hydrogen bonds interactions with NADP+ per monomer, 16 of which are mediated by water (Table S2). Notably, all of the residues involved directly NADP+ binding in E. coli SSADH [48] (Table S2) are also conserved in human SSADH.
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pone-0009280-g006: NADP+ binding of E. coli SSADH.Stereo view of the active site showing the NADP+ moiety (yellow), SSADH residues (green) involved in binding NADP+, water molecules can be seen as red spheres and all bonds are depicted with a black dashed line. The 2F0–Fc omit electron density of the NADP+ moiety contoured at 1σ is also shown (light blue mesh). Interactions of monomer A and NADP+ can be seen, specifically both AN1 and AN6 of the adenine moiety (labelled adn) interacts with Q239(Oε1), Q243(Oε1) and N217(Oδ1) via water molecules. Adjacent to the adenine moiety, both AO2 and AO3 of the ribose (labelled rb2) hydrogen bonds with T153(O) and K179(NZ, O) via a single water molecule. 3AOP of the 2'-phosphate interacts with K179(NZ). AO2 of the pyrophosphate interacts with S233(N, OG) and the NO1 hydrogen bonds directly with W155(Nε1). Both NO2 and NO3 of the adjacent ribose moiety (labelled rb1) hydrogen bonds with K338(NZ), while NO2 also interacts with E385(Oε1). NN7 of the nicotinamide (labelled nt) moiety interacts with N156(Nδ2) and the catalytic C288(N) via the single water molecule. While NO7 interacts directly with G232(O) and L255(O), as well as with L255(N) and E254(Oε1) via the same water molecule. Up to 13 SSADH residues make 24 van der Waals or hydrogen bonds interactions with NADP+ per monomer, 16 of which are mediated by water (Table S2). Notably, all of the residues involved directly NADP+ binding in E. coli SSADH [48] (Table S2) are also conserved in human SSADH.

Mentions: In the E. coli SSADH structure, electron density for the cofactor in the binding sites is clearly evident (Figure 6). Surface representation of the cofactor binding site illustrates where the cofactor is positioned; also shown is the human structure (PDB ID: 2w8r)[26] in which the cofactor NAD+ is soaked into the binding site of the C340A mutant (Figure 4A, B). The cofactor binding site comprises two pockets; one of which is close to the surface of the SSADH molecule and accommodates the adenosine (adenine and the first ribose) and the 2'phosphate. The second pocket is located centrally in the active site and accommodates the second ribose and the nicotinamide.


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)

NADP+ binding of E. coli SSADH.Stereo view of the active site showing the NADP+ moiety (yellow), SSADH residues (green) involved in binding NADP+, water molecules can be seen as red spheres and all bonds are depicted with a black dashed line. The 2F0–Fc omit electron density of the NADP+ moiety contoured at 1σ is also shown (light blue mesh). Interactions of monomer A and NADP+ can be seen, specifically both AN1 and AN6 of the adenine moiety (labelled adn) interacts with Q239(Oε1), Q243(Oε1) and N217(Oδ1) via water molecules. Adjacent to the adenine moiety, both AO2 and AO3 of the ribose (labelled rb2) hydrogen bonds with T153(O) and K179(NZ, O) via a single water molecule. 3AOP of the 2'-phosphate interacts with K179(NZ). AO2 of the pyrophosphate interacts with S233(N, OG) and the NO1 hydrogen bonds directly with W155(Nε1). Both NO2 and NO3 of the adjacent ribose moiety (labelled rb1) hydrogen bonds with K338(NZ), while NO2 also interacts with E385(Oε1). NN7 of the nicotinamide (labelled nt) moiety interacts with N156(Nδ2) and the catalytic C288(N) via the single water molecule. While NO7 interacts directly with G232(O) and L255(O), as well as with L255(N) and E254(Oε1) via the same water molecule. Up to 13 SSADH residues make 24 van der Waals or hydrogen bonds interactions with NADP+ per monomer, 16 of which are mediated by water (Table S2). Notably, all of the residues involved directly NADP+ binding in E. coli SSADH [48] (Table S2) are also conserved in human SSADH.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2823781&req=5

pone-0009280-g006: NADP+ binding of E. coli SSADH.Stereo view of the active site showing the NADP+ moiety (yellow), SSADH residues (green) involved in binding NADP+, water molecules can be seen as red spheres and all bonds are depicted with a black dashed line. The 2F0–Fc omit electron density of the NADP+ moiety contoured at 1σ is also shown (light blue mesh). Interactions of monomer A and NADP+ can be seen, specifically both AN1 and AN6 of the adenine moiety (labelled adn) interacts with Q239(Oε1), Q243(Oε1) and N217(Oδ1) via water molecules. Adjacent to the adenine moiety, both AO2 and AO3 of the ribose (labelled rb2) hydrogen bonds with T153(O) and K179(NZ, O) via a single water molecule. 3AOP of the 2'-phosphate interacts with K179(NZ). AO2 of the pyrophosphate interacts with S233(N, OG) and the NO1 hydrogen bonds directly with W155(Nε1). Both NO2 and NO3 of the adjacent ribose moiety (labelled rb1) hydrogen bonds with K338(NZ), while NO2 also interacts with E385(Oε1). NN7 of the nicotinamide (labelled nt) moiety interacts with N156(Nδ2) and the catalytic C288(N) via the single water molecule. While NO7 interacts directly with G232(O) and L255(O), as well as with L255(N) and E254(Oε1) via the same water molecule. Up to 13 SSADH residues make 24 van der Waals or hydrogen bonds interactions with NADP+ per monomer, 16 of which are mediated by water (Table S2). Notably, all of the residues involved directly NADP+ binding in E. coli SSADH [48] (Table S2) are also conserved in human SSADH.
Mentions: In the E. coli SSADH structure, electron density for the cofactor in the binding sites is clearly evident (Figure 6). Surface representation of the cofactor binding site illustrates where the cofactor is positioned; also shown is the human structure (PDB ID: 2w8r)[26] in which the cofactor NAD+ is soaked into the binding site of the C340A mutant (Figure 4A, B). The cofactor binding site comprises two pockets; one of which is close to the surface of the SSADH molecule and accommodates the adenosine (adenine and the first ribose) and the 2'phosphate. The second pocket is located centrally in the active site and accommodates the second ribose and the nicotinamide.

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