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

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IUBMB Comments
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+ .
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UNIPROT: B1XMM6
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The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
aldh21, sp2771, syssadh, abssadh, apssadh, all3556, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
succinic semialdehyde dehydrogenase
-
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].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
show the reaction diagram
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
succinate semialdehyde + NADP+ + H2O
succinate + NADPH + 2 H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
NADPH
binding structure, overview
additional information
no detectable activity by using NAD+ as cofactor
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
H2O2
50 microM H2O2 sharply reduces activity to 31% of the H2O2-free enzyme and activity further decreases to 3% at 1 mM H2O2
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DTT
enzyme activity increases significantly, and the enzyme becomes resistant to oxidative stress in presence of NADP+ and DTT
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.439
NADP+
30°C, pH 7.6
additional information
additional information
the Km-value of succinate semialdehyde estimated to be far less than 0.05 mM
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5
NADP+
30°C, pH 7.6
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39.6
purified recombinant enzyme, pH 7.6, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
SSADH belongs to the aldehyde dehydrogenase (ALDH) superfamily
metabolism
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
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
B1XMM6_SYNP2
Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6)
454
0
49019
TrEMBL
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
91000
analytical ultracentrifugation
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
Sp2771 monomer is composed of N-terminal cofactor binding domain, a catalytic domain and an oligomerization domain
homodimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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 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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Park, J.; Rhee, S.
Structural basis for a cofactor-dependent oxidation protection and catalysis of cyanobacterial succinic semialdehyde dehydrogenase
J. Biol. Chem.
288
15760-15770
2013
Synechococcus sp. (B1XMM6)
Manually annotated by BRENDA team
Yuan, Z.; Yin, B.; Wei, D.; Yuan, Y.R.
Structural basis for cofactor and substrate selection by cyanobacterium succinic semialdehyde dehydrogenase
J. Struct. Biol.
182
125-135
2013
Synechococcus sp. (B1XMM6)
Manually annotated by BRENDA team
Park, J.; Rhee, S.
Structural basis for a cofactor-dependent oxidation protection and catalysis of cyanobacterial succinic semialdehyde dehydrogenase
J. Biol. Chem.
288
15760-15770
2013
Synechococcus sp. (B1XMM6), Synechococcus sp. ATCC 27264 (B1XMM6)
Manually annotated by BRENDA team