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Enzyme Nomenclature
Information on EC 1.2.1.79 - succinate-semialdehyde dehydrogenase (NADP+)
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
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EC NUMBER 
COMMENTARY 
1.2.1.79
-
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RECOMMENDED NAME
GeneOntology No.
succinate-semialdehyde dehydrogenase (NADP+)
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
succinate semialdehyde + NADP+ + H2O = succinate + NADPH + 2 H+
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
succinate-semialdehyde:NADP+ oxidoreductase
This enzyme participates in the degradation of glutamate and 4-aminobutyrate.
It is similar to EC 1.2.1.24 [succinate-semialdehyde dehydrogenase (NAD+)], and EC 1.2.1.16 [succinate-semialdehyde dehydrogenase (NAD(P)+)], but is specific for NADP+. The enzyme from Escherichia coli is 20-fold more active with NADP+ than NAD+ [2].
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
metabolism
physiological function
additional information
evolution
-
SSADH belongs to the aldehyde dehydrogenase (ALDH) superfamily
-
metabolism
-
SSADH plays an essential role in the metabolism of the inhibitory neurotransmitter c-aminobutyric acid
metabolism
enzyme catalyzes the last step of the gamma-aminobutyrate degradation
metabolism
-
succinic semialdehyde dehydrogenase from Synechococcus is an essential enzyme in the tricarboxylic acid cycle of cyanobacteria
metabolism
-
Sp2771 enzyme completes together with a novel 2-oxoglutarate decarboxylase a non-canonical tricarboxylic acid cycle
metabolism
-
SSADH plays an essential role in the metabolism of the inhibitory neurotransmitter c-aminobutyric acid
-
metabolism
-
enzyme catalyzes the last step of the gamma-aminobutyrate degradation
-
physiological function
gene is disrupted in a transposon-induced mutant of Ralstonia eutropha exhibiting the phenotype 4-hydroxybutyric acid-leaky
physiological function
-
succinic semialdehyde dehydrogenase from Synechococcus is an essential enzyme in the tricarboxylic acid, TCA, cycle of cyanobacteria. It completes a 2-oxoglutarate dehydrogenase-deficient cyanobacterial TCA cycle through a detour metabolic pathway. SySSADH produces succinate in an NADP+ -dependent manner with a single cysteine acting as the catalytic residue in the catalytic loop
physiological function
-
succinic semialdehyde dehydrogenase from Synechococcus is an essential enzyme in the tricarboxylic acid, TCA, cycle of cyanobacteria. It completes a 2-oxoglutarate dehydrogenase-deficient cyanobacterial TCA cycle through a detour metabolic pathway. SySSADH produces succinate in an NADP+ -dependent manner with a single cysteine acting as the catalytic residue in the catalytic loop
-
additional information
-
structure analysis of the enzyme in binary and ternary with NADP(H) and/or substrate reveals that the enzyme forms a distinct reaction intermediate in each complex: a covalent adduct of a cofactor with the catalytic cysteine in the binary complex and a proposed thiohemiacetal intermediate in the ternary complex. SySSADH produces succinate in an NADP+ -dependent manner with a single cysteine acting as the catalytic residue in the catalytic loop, catalytic mechanism, overview. The formation of the NADP-cysteine adduct is a kinetically preferred event that protects the catalytic cysteine from H2O2-dependent oxidative stress. SySSADH shows a cofactor-dependent oxidation protection in 1-Cys SSADH, which is unique relative to other 2-Cys SSADHs employing a redox-dependent formation of a disulfide bridge. The catalytic cysteine preferentially forms an NADP-cysteine adduct if NADP+ is present
additional information
Ser157 residue in Sp2771 plays a critical structural role in determining NADP+ preference for Sp2771, whereas size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection. Enzyme Sp2771 structure modelling comprising residues 2-454, active site and substrate binding structures, overview
additional information
-
structure analysis of the enzyme in binary and ternary with NADP(H) and/or substrate reveals that the enzyme forms a distinct reaction intermediate in each complex: a covalent adduct of a cofactor with the catalytic cysteine in the binary complex and a proposed thiohemiacetal intermediate in the ternary complex. SySSADH produces succinate in an NADP+ -dependent manner with a single cysteine acting as the catalytic residue in the catalytic loop, catalytic mechanism, overview. The formation of the NADP-cysteine adduct is a kinetically preferred event that protects the catalytic cysteine from H2O2-dependent oxidative stress. SySSADH shows a cofactor-dependent oxidation protection in 1-Cys SSADH, which is unique relative to other 2-Cys SSADHs employing a redox-dependent formation of a disulfide bridge. The catalytic cysteine preferentially forms an NADP-cysteine adduct if NADP+ is present
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
3-carboxybenzaldehyde + NADP+ + H2O
3-carboxybenzoate + NADPH + 2 H+
-
-
-
?
3-nitrobenzaldehyde + NADP+ + H2O
3-nitrobenzoate + NADPH + 2 H+
-
-
-
?
4-carboxybenzaldehyde + NADP+ + H2O
4-carboxybenzoate + NADPH + H+
-
-
-
?
malonate semialdehyde + NADP+ + H2O
malonate + NADPH + 2 H+
-
8.7% of the activity with succinate semialdehyde
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + 2 H+
about 11% of the activity with NADP+
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + 2 H+
-
-
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + 2 H+
kcat/Km for NADP+ is 250fold higher compared to kcat/Km for NAD+
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + 2 H+
kcat/Km for NADP+ is 250fold higher compared to kcat/Km for NAD+
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
-
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
kcat/Km for NADP+ is 250fold higher compared to kcat/Km for NAD+
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
kcat/Km for NADP+ is 250fold higher compared to kcat/Km for NAD+
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
data from crystal structures provide details about the catalytic mechanism by revealing a covalent adduct of a cofactor with the catalytic cysteine in the binary complex and a proposed thiohemiacetal intermediate in the ternary complex
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
binding structure, overview
-
-
r
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
r
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
binding structure, overview
-
-
r
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + H+
-
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + H+
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinic semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
chemical mechanism based on functional data and structural information proposed, 1H-NMR to probe the stereospecificity of GabD1 show a transfer of the deuteride to the pro-R position of NADP+ indicating GabD1 has A-type stereospecificity
-
-
ir
succinic semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
chemical mechanism based on functional data and structural information proposed, 1H-NMR to probe the stereospecificity of GabD1 show a transfer of the deuteride to the pro-R position of NADP+ indicating GabD1 has A-type stereospecificity
-
-
ir
additional information
?
-
only the aldehyde forms and not the gem-diol forms of the specific substrate succinic semialdehyde , of selected aldehyde substrates, and of the inhibitor 3-tolualdehyde bind to the enzyme
-
-
-
additional information
?
-
-
no substrate: n-butanal, formaldehyde, acetaldehyde, glyoxal, glyoxalate, propanal, glutaraldehyde, benzaldehyde, and anisaldehyde
-
-
-
additional information
?
-
-
no substrate: glyoxylic acid, formic acid, formaldehyde, acetaldehyde, glyoxal, furfural and acrolein
-
-
-
additional information
?
-
other aldehydes, such as formaldehyde, acetaldehyde and glutaraldehyde, are very poor substrates showing a narrow substrate specificity of GabD1
-
-
?
additional information
?
-
other aldehydes, such as formaldehyde, acetaldehyde and glutaraldehyde, are very poor substrates showing a narrow substrate specificity of GabD1
-
-
?
additional information
?
-
-
in the binary enzyme-succinate semialdehyde-complex of SpSSADH, the succinate semialdehyde shows a tightly bound bent form nearby the catalytic residues, which may be caused by reduction of the cavity volume for substrate binding, compared with other SSADHs. Structural comparison of the tertiary enzyme-succinate semialdehyde-NADP+-complex with a binary complex + of SpSSADH with NADP indicates that the substrate inhibition is induced by the binding of inhibitory succinate semialdehyde in the cofactor-binding site, instead of NADP+. In the active site of enzyme SpSSADH, SSA is buried inside of the substrate-binding pocket formed by Phe133, Tyr136, Val262, Trp418 and Phe426 residues, substrate binding structure, overview
-
-
-
additional information
?
-
in the binary enzyme-succinate semialdehyde-complex of SpSSADH, the succinate semialdehyde shows a tightly bound bent form nearby the catalytic residues, which may be caused by reduction of the cavity volume for substrate binding, compared with other SSADHs. Structural comparison of the tertiary enzyme-succinate semialdehyde-NADP+-complex with a binary complex + of SpSSADH with NADP indicates that the substrate inhibition is induced by the binding of inhibitory succinate semialdehyde in the cofactor-binding site, instead of NADP+. In the active site of enzyme SpSSADH, SSA is buried inside of the substrate-binding pocket formed by Phe133, Tyr136, Val262, Trp418 and Phe426 residues, substrate binding structure, overview
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
-
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinate semialdehyde + NAD+ + H2O
succinate + NADH + H+
P25526
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
-
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
A0A0J9X1M8
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
B1XMM6
data from crystal structures provide details about the catalytic mechanism by revealing a covalent adduct of a cofactor with the catalytic cysteine in the binary complex and a proposed thiohemiacetal intermediate in the ternary complex
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
B1XMM6
-
-
-
r
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
B1XMM6
-
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + H+
-
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + H+
P25526
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
-
-
?
succinic semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
P9WNX9
chemical mechanism based on functional data and structural information proposed, 1H-NMR to probe the stereospecificity of GabD1 show a transfer of the deuteride to the pro-R position of NADP+ indicating GabD1 has A-type stereospecificity
-
-
ir
succinic semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
P9WNX9
chemical mechanism based on functional data and structural information proposed, 1H-NMR to probe the stereospecificity of GabD1 show a transfer of the deuteride to the pro-R position of NADP+ indicating GabD1 has A-type stereospecificity
-
-
ir
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD+
-
NAD+ acts as cosubstrate, but the reaction rates are more than 20fold lower than those with NADP+
NAD+
-
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
NAD+
kcat/Km for NADP+ is 250fold higher compared to kcat/Km for NAD+
NADP+
-
NAD+ also acts as cosubstrate, but the reaction rates are more than 20fold lower than those with NADP+
NADP+
-
the enzyme activity in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
NADP+
electron density analysis of binding site. The enzyme activity measured in the presence of NADP+ is approximately 20fold higher than that measured in the presence of NAD+
NADP+
kcat/Km for NADP+ is 250fold higher compared to kcat/Km for NAD+
NADP+
-
enzyme activity increases significantly, and the enzyme becomes resistant to oxidative stress in presence of NADP+ and DTT
NADP+
Ser157 residue in Sp2771 plays a critical structural role in determining NADP+ preference for Sp2771, whereas size and distribution of hydrophobic residues along the substrate binding funnel determine substrate selection
additional information
-
no detectable activity by using NAD+ as cofactor
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3-tolualdehyde
-
only the aldehyde forms and not the gem-diol forms of the inhibitor 3-tolualdehyde bind to the enzyme
H2O2
-
50 microM H2O2 sharply reduces activity to 31% of the H2O2-free enzyme and activity further decreases to 3% at 1 mM H2O2
Succinic semialdehyde
partial substrate inhibition because velocity decreases to a non-zero value at saturating concentrations of Mg2+ and succinic semialdehyde
succinate semialdehyde
-
uncompetitive substrate inhibition above 0.02 mM, in the presence of NADP+. Substrate inhibition is induced by the binding of inhibitory succinate semialdehyde in the cofactor-binding site, instead of NADP+
succinate semialdehyde
-
complete uncompetitive substrate inhibition. Structural comparison of the tertiary enzyme-succinate semialdehyde-NADP+-complex with a binary complex of SpSSADH with NADP indicates that the substrate inhibition is induced by the binding of inhibitory succinate semialdehyde in the cofactor-binding site, instead of NADP+
additional information
-
analysis of the kinetic inhibitory parameters revealed significant substrate inhibition in the presence of NADP+ at concentrations of succinate semialdehyde higher than 0.02 mM, which exhibits complete uncompetitive substrate inhibition, structure-based molecular insights into the substrate inhibition mechanism of SpSSADH, overview
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
DTT
-
enzyme activity increases significantly, and the enzyme becomes resistant to oxidative stress in presence of NADP+ and DTT
2-mercaptoethanol
-
loss of about 80% of original acitivity after exhaustive dialysis, renaturation in presence of 2-mercaptoethanol
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0092
NADP+
steady-state parameter, 50 microM NADP+ and 10 mM Mg2+, at pH 7.5 and 37°C
0.0613
NADP+
steady-state parameter, 50 microM NADP+, at pH 7.5 and 37°C
0.01694
succinate semialdehyde
-
in 100 mM sodium phosphate buffer, pH 8.0, 30°C
0.003
Succinic semialdehyde
steady-state parameter, 200 microM succinic semialdehyde and 10 mM Mg2+, at pH 7.5 and 37°C
0.0133
Succinic semialdehyde
steady-state parameter, 200 microM succinic semialdehyde, at pH 7.5 and 37°C
additional information
additional information
enzyme does not obey Michaelis-Menten kinetics
-
additional information
additional information
-
the Km-value of succinate semialdehyde estimated to be far less than 0.05 mM
-
additional information
additional information
-
Michaelis-Menten kinetics
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.3
NADP+
steady-state parameter, 50 microM NADP+, at pH 7.5 and 37°C
2.7
NADP+
steady-state parameter, 50 microM NADP+ and 10 mM Mg2+, at pH 7.5 and 37°C
1.9
Succinic semialdehyde
steady-state parameter, 200 microM succinic semialdehyde, at pH 7.5 and 37°C
4.7
Succinic semialdehyde
steady-state parameter, 200 microM succinic semialdehyde and 10 mM Mg2+, at pH 7.5 and 37°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
21
NADP+
steady-state parameter, 50 microM NADP+, at pH 7.5 and 37°C
290
NADP+
steady-state parameter, 50 microM NADP+ and 10 mM Mg2+, at pH 7.5 and 37°C
140
Succinic semialdehyde
steady-state parameter, 200 microM succinic semialdehyde, at pH 7.5 and 37°C
1600
Succinic semialdehyde
steady-state parameter, 200 microM succinic semialdehyde and 10 mM Mg2+, at pH 7.5 and 37°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0628
Succinic semialdehyde
partial substrate inhibition, pH 7.5 and 37°C
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.5 - 10
GabD1 could not be assayed below pH 5.5, due to precipitation and loss of activity
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
22
30
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Streptococcus pyogenes MGAS1882;
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
-
Sp2771 monomer is composed of N-terminal cofactor binding domain, a catalytic domain and an oligomerization domain
homodimer
tetramer
additional information
the central parts of both cofactor binding domain and catalytic domain contain atypical alpha/beta structure. The catalytic domain consists of a seven stranded beta-sheet flanked by two alpha-helices on one side and three alpha-helices on the other side.The cofactor binding domain displays the two tandem Rossmann folds for NADP+ binding . The oligomerization domain contains three-stranded antiparallel beta-sheets protruding across the center of the dimer interface, while the NADP+ binding site is on the opposite side of the dimer interface
homodimer
2 * 50000, SDS-PAGE; 2 * 51600, calculated from sequence
homodimer
-
gel filtration, SySSADH monomer consists of three segments - alpha/beta-fold N- and C-domains for a cofactor binding and a catalytic domain, and three antiparallel beta-strands constituting a dimerization domain
homodimer
-
the overall structure of monomeric SySSADH is reminiscent of an ALDH fold. It is made up of three segments: alpha/beta-fold N- and C-domains for a cofactor binding and a catalytic domain, respectively, and three antiparallel beta-strands constituting a dimerization domain
homodimer
-
the overall structure of monomeric SySSADH is reminiscent of an ALDH fold. It is made up of three segments: alpha/beta-fold N- and C-domains for a cofactor binding and a catalytic domain, respectively, and three antiparallel beta-strands constituting a dimerization domain
-
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with NADP+, hanging drop vapour diffusion method, using 0.2 M ammonium tartrate, 26-31% polyethylene glycol 3350, 10 mM beta-mercaptoethanol and 0.1 M Tris (pH 7.2-7.5)
-
to 1.4 A resolution. The overall structure of SSADH shares the general fold of ALDH classes 1 and 2. The SSADH monomer is composed of three domains; an N-terminal NAD(P)-binding domain of residues 1–125, 148–256, and 457–472, a catalytic domain of residues 257–456, and an oligomerization domain of residues 126–147 and 473–482. The catalytic loop of Escherichia coli SSADH, unlike that of human SSADH, does not undergo disulfide bond-mediated structural changes upon changes of environmental redox status. The protein is not regulated via redox-switch modulation. A difference in the conformation of the connecting loop beta15–beta16 causes the formation of a water molecule-mediated hydrogen bond network between the connecting loop and the catalytic loop in Escherichia coli SSADH
-
comparison to human enzyme, analysis of NADP+ binding site. Enzyme is a homotetramer with the 4 monomers related by a non-crystallographic 222 symmetry. The conserved catalytic site residues and active site residues correspond to C288 and E254 as well as R164, R282 and S445, respectively
enzyme SpSSADH in a binary complex with succinate semialdehyde as the substrate and a ternary complex with succinate semialdehyde and NADP+, hanging-drop vapor diffusion method, mixing of mixture of 0.001 ml of protein solution with 0.001 ml of reservoir solution, for the binary complex crystal, SpSSADH is pre-incubated with succinate semialdehyde at the molar ratio of 1:2, and the protein-substrate mixture is crystallized over 00.5 ml of reservoir solution containing 0.1 M sodium acetate trihydrate, pH 4.6, and 2.0 M ammonium sulfate, the trinary complex is obtained by soaking the pre-grown NADP+ co-crystallized crystal with a 1:10 molar ratio of succinate semialdehyde under the same reservoir conditions, 22°C, X-ray diffraction structure determination and analysis at 2.4 A resolution, molecular replacement method with the apo-structure of SpSSADH, PDB ID 4OGD, as the search model
-
structures in a binary complex with succinic semialdehyde as the substrate and a ternary complex with the substrate succinic semialdehyde and the inhibitory succinate semialdehyde, at 2.4 A resolution for both structures
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structures in apo-form and in a binary complex with NADP+ at 1.6 A and 2.1 A resolutions, respectively. Both structures show dimeric conformation and contain a single cysteine residue in the catalytic loop of each subunit. Residues Ser158 and Tyr188 participate in the stabilization of the 2'-phosphate group of adenine-side ribose in NADP+
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crystal structures of SySSADH determined in their apo form, as a binary complex with NADP+ and as a ternary complex with succinic semialdehyde and NADPH, resoultion of 1.7 A for the apo form and of 1.4 A for the binary and ternary complex
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crystal structures of wild type Sp2771 at 2.1 A resolution, Sp2771 S419A mutant at 2.5 A resolution and ternary structure of non-catalytic Sp2771 C262A mutant in complex with NADP + and succinate semialdehyde at 1.7 A resolution
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purified recombinant enzyme in apo form, in a binary complex with NADP+, and in a ternary complex with succinic semialdehyde and NADPH, sitting drop vapor diffusion method, using a crystallization buffer of 0.05 M potassium phosphate monobasic, 20% w/v PEG 8000, and 2 mM CaCl2, 22°C, for the binary and tertiary complexes, a pre-grown crystals of SySSADH are soaked for 60 min in a solution of 0.05 M potassium phosphate monobasic, 20% w/v PEG8000, 30% v/v ethylene glycol, and 50 mM NADPH or 50 mM NADPH and 50 mM succinate semialdehyde, respectively, X-ray diffraction structure determination and analysis at 1.4-1.7 A resolution, single-wavelength diffraction, modelling
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purified recombinant His6-tagged wild-type Sp2771, and Sp2771 S419A and Sp2771 C262A mutants in ternary complex with NADP+ and succinate semialdehyde, mixing of 0.001 ml of protein solution and reservoir solution each, the latter containing 15% PEG 5000 MME , 1 mM DTT, 3% tascimate, and 100 mM HEPES, pH 6.8 for the wild-type enzyme and the mutants, for Sp2771 mutants in complex with NADP+ and succinate semialdehyde, NAD+ and succinate semialdehyde are incubated with proteins for 15 min at room temperature before crystallization, 20°C, 3 days, X-ray diffraction structure determination and analysis at 1.7-2.5 A resolution, molecular replacement using Escherichia coli SSADH structure, PDB ID 3JZ4, as search model
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
SySSADH is an oxidation-sensitive enzyme and the formation of the NADP-cysteine adduct is a kinetically preferred event that protects the catalytic Cys-262 from H2O2-dependent oxidative stress
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725523
SySSADH is an oxidation-sensitive enzyme, the formation of the NADP-cysteine adduct is a kinetically preferred event that protects the catalytic cysteine from H2O2-dependent oxidative stress. Over 70% of the original SySSADH activity is maintained with 0.005-0.25 mM H2O2 with a further drop of activity to 40% at 1 m H2O2. Comparable or even higher enzyme activity is observed when SySSADH is preincubated for 10 min with 2.5 mM NADP+ followed by H2O2 treatment for 60 min
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742844
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nickel chelating Sepharose column chromatography and S200 16/60 gel filtration
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recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, and ultrafiltration
recombinant N-terminally His-tagged SySSADH from Escherichia coli strain B834(DE3) by nickel affinity chromatography, dialysis and cleavge fo the His-tag by tobacco etch mosaic virus protease, followed by another step of immobilized metal affinity chromatography and gel filtration. Recombinant His-tagged enzyme mutants from Escherichia coli strain coli BL21(DE3) by immobilized metal affinity chromatography and dialysis
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through Ni2+ affinity column chromatography, followed by a Hi-Load Superdex S-75 26/60 column chromatography
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using immobilized metal affinity chromatography, removal of N-terminal His tag, further purification by immobilized metal affinity chromatography and gel filtration using a Superdex 200 column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
gene ssadh, recombinant expression in Escherichia coli strain BL21(DE3)
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insertion of the full length Sp2771 gene into pET28b vector with an N-terminal His-6 tag and expression in Escherichia coli (BL21/DE3) strain
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N-terminal His-tagged SySSADH expressed in Escherichia coli B834 (DE3) methionine auxotroph cells
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recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21/DE3
recombinant expression of N-terminally His-tagged SySSADH (residues Met1 to Lys454) in Escherichia coli methionine-auxotrophic strain B834(DE3), recombinant expression of His-tagged enzyme mutants in Escherichia coli strain coli BL21(DE3). Juxtaposition of the N- and C-domains generates an active site tunnel between the two domains that is accessible from both ends. The catalytic residues are located in the middle of the tunnel. A nucleophile Cys262 is located in the catalytic loop between alphaa8 and beta11 of the C-domain, and a general base Glu228 is located in an interdomain-connecting loop between beta9 and beta10. Dimerization is mediated largely by N-domain alpha7 and the three antiparallel beta-strands (beta3, beta4, beta17) protruding from the N- and C-domains
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induction is highly coordinated with putrescine:2-oxoglutarate transaminase, gamma-aminobutyraldehyde dehydrogenase and gamma-aminobutyrate:2-oxoglutarate transaminase activities
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the expression of gabD is not specifically induced by either 4-hydroxybutanoic acid or gamma-aminobutanoate
the genes gabT, coding for glutamate:succinic semialdehyde transaminase, and gabD, encoding succinic semialdehyde dehydrogenase, are cotranscribed from a promoter located upstream of gabD
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C262A
E228A
E228Q
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active site mutation, nonfunctional because Glu-228 acts as a general base
F132A
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activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity
F425A
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inactive, suggesting that Phe-425 plays an important role in substrate binding
I263A
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activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity
N131A
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mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity
N131D
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mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity
R139A
R139K
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mutant enzyme exhibited an activity up to 80% that of the wild type enzyme, suggesting the significance of a positively charged residue in the binding of the carboxyl group of succinic semialdehyde
S157E
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mutation changes cofactor preference from NADP+ to NAD+, but enzyme activity is approximately 10fold reduced
S419A
W135A
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activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity
C262A
site-directed mutagenesis, Sp2771 mutant structure analysis and comparison to the wild-type structure
S419A
site-directed mutagenesis, Sp2771 mutant structure analysis and comparison to the wild-type structure
C262A
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active site mutation, nonfunctional because Cys-262 acts as a nucleophilel
E228A
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active site mutation, nonfunctional because Glu-228 acts as a general base
R139A
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activity of about 10–30% of the wild type enzyme, indicating a contribution of these succinic semialdehyde binding residues to the overall enzyme activity
R139A
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90% reduced catalytic activity, residue is involved in substrate binding
S419A
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mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity
S419A
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80% reduced catalytic activity, residue is involved in substrate binding
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
analysis of Escherichia coli SSADH structure with respect to human disease-linked mutations
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LINKS TO OTHER DATABASES (specific for EC-Number 1.2.1.79)
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