Information on EC 1.2.1.79 - succinate-semialdehyde dehydrogenase (NADP+)

GabD1

NADP+-dependent succinic semialdehyde dehydrogenase

Sp2771

SpSSADH

SSADH

SSADH-II

SSO1842

succinic semialdehy de dehydrogenase

-

succinic semialdehyde dehydrogenase

SYNPCC7002_A2771

gene name, UniProt

SySSADH

glutamate and glutamine metabolism

succinate semialdehyde + NADP+ + H2O = succinate + NADPH + 2 H+

-

BRENDA

KEGG

Alanine, aspartate and glutamate metabolism, Butanoate metabolism, Nicotinate and nicotinamide metabolism

MetaCyc

4-aminobutanoate degradation II, 4-hydroxyphenylacetate degradation, nicotine degradation I (pyridine pathway), nicotine degradation II (pyrrolidine pathway), TCA cycle IV (2-oxoglutarate decarboxylase)

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].

2,5-dioxopentanoate + NAD+ + H2O

?

2,5-dioxopentanoate + NADP+ + H2O

?

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+

-

-

?

benzaldehyde + NADP+ + H2O

benzoate + NADPH + 2 H+

-

-

?

malonate semialdehyde + NADP+ + H2O

malonate + NADPH + 2 H+

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8.7% of the activity with succinate semialdehyde

-

?

n-butanal + NADP+ + H2O

butanoate + NADPH + 2 H+

-

-

?

n-hexanal + NADP+ + H2O

hexanoate + NADPH + H+

-

-

?

n-pentanal + NADP+ + H2O

n-pentanoate + NADPH + H+

-

-

?

succinate semialdehyde + NAD+ + H2O

succinate + NADH + 2 H+

5 entries

succinate semialdehyde + NAD+ + H2O

succinate + NADH + H+

succinate semialdehyde + NADP+ + H2O

succinate + NADPH + 2 H+

20 entries

succinate semialdehyde + NADP+ + H2O

succinate + NADPH + H+

succinic semialdehyde + NADP+ + H2O

succinate + NADPH + 2 H+

additional information

?

-

succinate semialdehyde + NAD+ + H2O

succinate + NADH + H+

succinate semialdehyde + NADP+ + H2O

succinate + NADPH + 2 H+

succinate semialdehyde + NADP+ + H2O

succinate + NADPH + H+

succinic semialdehyde + NADP+ + H2O

succinate + NADPH + 2 H+

NAD+

NADP+

NADPH

binding structure, overview

additional information

-

Ca2+

2 to 3-fold activation at saturating concentration

Co2+

2 to 3-fold activation at saturating concentration

K+

-

5 mM, 10% activation

Mg2+

2 to 3-fold activation at saturating concentration

Mn2+

2 to 3-fold activation at saturating concentration

Ni2+

2 to 3-fold activation at saturating concentration

additional information

no activation with Cd2+ or Zn2+

3-tolualdehyde

only the aldehyde forms and not the gem-diol forms of the inhibitor 3-tolualdehyde bind to the enzyme

5,5'-dithiobis(2-nitrobenzoic acid)

-

0.01 mM, 29% residual activity

Ca2+

-

H2O2

50 microM H2O2 sharply reduces activity to 31% of the H2O2-free enzyme and activity further decreases to 3% at 1 mM H2O2

Mg2+

-

Mn2+

-

N-ethylmaleimide

-

0.1 mM, complete loss of activity

NADPH

Ni2+

-

succinate semialdehyde

Succinic semialdehyde

partial substrate inhibition because velocity decreases to a non-zero value at saturating concentrations of Mg2+ and succinic semialdehyde

Zn2+

-

1 mM, complete inhibition

additional information

-

2-mercaptoethanol

dithiothreitol

DTT

enzyme activity increases significantly, and the enzyme becomes resistant to oxidative stress in presence of NADP+ and DTT

0.1 - 0.2

2,5-dioxopentanoate

0.23 - 11.1

NAD+

0.0092 - 0.439

NADP+

10 entries

0.001 - 3.95

succinate semialdehyde

11 entries

0.003 - 0.0133

Succinic semialdehyde

additional information

-

21.2 - 27.3

2,5-dioxopentanoate

1.94 - 136.3

NAD+

1.3 - 16.5

NADP+

5 entries

1.69 - 18.4

succinate semialdehyde

4 entries

1.9 - 4.7

Succinic semialdehyde

134.1 - 215.3

2,5-dioxopentanoate

9.7 - 12.3

NAD+

7.2 - 3066

NADP+

4 entries

0.43 - 3454

succinate semialdehyde

4 entries

140 - 1600

Succinic semialdehyde

0.1

succinate semialdehyde

0.0628

Succinic semialdehyde

partial substrate inhibition, pH 7.5 and 37°C

0.08

3-tolualdehyde

Escherichia coli

pH 8.5, 22°C

132.1

cosubstrate NADP+, pH 11.0, 30°C

14.9

Cupriavidus necator

Q9RBF6

cosubstrate NAD+, pH 11.0, 30°C

34

pH 8.5, 22°C

39.6

purified recombinant enzyme, pH 7.6, 30°C

4.1

-

pH 7.8, 30°C

288038

7.9

-

pH 8.7, 35°C

11

-

6.5

assay at

7

assay at

7.5 - 7.8

-

7.6

assay at

8.5

-

8.7

-

9

-

5.5 - 10

GabD1 could not be assayed below pH 5.5, due to precipitation and loss of activity

7.4

75% of maximum activity

22

30

31

-

35 - 45

-

37

-

assay at

70

assay at

-

UniProt

Escherichia coli MC1061

-

UniProt

-

-

-

288038

-

-

Mycobacterium tuberculosis H37Rv

UniProt

-

UniProt

-

-

-

Saccharolobus solfataricus P2

UniProt

-

UniProt

-

739994, 741008, 741822

Streptococcus pyogenes MG-AS1882

UniProt

-

Streptococcus pyogenes MGAS1882

UniProt

-

739994, 741008

UniProt

-

725523, 725838, 742844

Synechococcus sp. ATCC 27264

UniProt

-

UniProt

-

UniProt

-

evolution

metabolism

physiological function

additional information

Streptococcus pyogenes MGAS1882

114000

gel filtration

200000

Pseudomonas putida

-

gel filtration

707583

300000

-

gel filtration

288038

50000

2 * 50000, SDS-PAGE

5022

x * 5022, calculated, x * 51000, SDS-PAGE

51000

Cupriavidus necator

Q9RBF6

x * 5022, calculated, x * 51000, SDS-PAGE

51600

2 * 51600, calculated from sequence

60000

-

x * 60000, calculated

708907

91000

analytical ultracentrifugation

?

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

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

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707392

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

739994

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+

741008

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

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

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

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

C262A

E228A

E228Q

active site mutation, nonfunctional because Glu-228 acts as a general base

F132A

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

inactive, suggesting that Phe-425 plays an important role in substrate binding

I263A

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

mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity

N131D

mutation of a residue that interacts with the O4 atom or the carboxyl group of succinic semialdehyde thus abolishing enzyme activity

R139A

R139K

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

mutation changes cofactor preference from NADP+ to NAD+, but enzyme activity is approximately 10fold reduced

S419A

W135A

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

8

-

rapid decrease in stability above

707580

18

-

14 h, 78% residual activity

37

-

14 h, 69% residual activity

50

10 min, stable

60

-

stable up to

GabD1 is naturally resistant to oxidation by H2O2

724115

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

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

742844

-15C, 14 h, 81% residual activity

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18C, 14 h, 78% residual activity

-

5°C, unstable unless 30% glycerol is added

-

-

nickel chelating Sepharose column chromatography and S200 16/60 gel filtration

recombinant enzyme

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

through Ni2+ affinity column chromatography, followed by a Hi-Load Superdex S-75 26/60 column chromatography

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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expressed in Escherichia coli BL21(DE3) cells

expression in Escherichia coli

gene ssadh, recombinant expression in Escherichia coli strain BL21(DE3)

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

N-terminal His-tagged SySSADH expressed in Escherichia coli B834 (DE3) methionine auxotroph cells

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

decrease by growth of cells on gamma-aminobutanoate

-

induction by growth on glutamate

-

induction is highly coordinated with putrescine:2-oxoglutarate transaminase, gamma-aminobutyraldehyde dehydrogenase and gamma-aminobutyrate:2-oxoglutarate transaminase activities

Pseudomonas putida

-

707583

the expression of gabD is not specifically induced by either 4-hydroxybutanoic acid or gamma-aminobutanoate

Cupriavidus necator

Q9RBF6

the genes gabT, coding for glutamate:succinic semialdehyde transaminase, and gabD, encoding succinic semialdehyde dehydrogenase, are cotranscribed from a promoter located upstream of gabD