Activating Compound | Comment | Organism | Structure |
---|---|---|---|
DTT | enzyme activity increases significantly, and the enzyme becomes resistant to oxidative stress in presence of NADP+ and DTT | Synechococcus sp. |
Cloned (Comment) | Organism |
---|---|
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 | Synechococcus sp. |
Crystallization (Comment) | Organism |
---|---|
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 | Synechococcus sp. |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinate semialdehyde + NADP+ + H2O | Synechococcus sp. | - |
succinate + NADPH + 2 H+ | - |
r | |
succinate semialdehyde + NADP+ + H2O | Synechococcus sp. ATCC 27264 | - |
succinate + NADPH + 2 H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Synechococcus sp. | B1XMM6 | - |
- |
Synechococcus sp. ATCC 27264 | B1XMM6 | - |
- |
Oxidation Stability | Organism |
---|---|
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 | Synechococcus sp. |
Purification (Comment) | Organism |
---|---|
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 | Synechococcus sp. |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
39.6 | - |
purified recombinant enzyme, pH 7.6, 30°C | Synechococcus sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
succinate semialdehyde + NADP+ + H2O | - |
Synechococcus sp. | succinate + NADPH + 2 H+ | - |
r | |
succinate semialdehyde + NADP+ + H2O | binding structure, overview | Synechococcus sp. | succinate + NADPH + 2 H+ | - |
r | |
succinate semialdehyde + NADP+ + H2O | - |
Synechococcus sp. ATCC 27264 | succinate + NADPH + 2 H+ | - |
r | |
succinate semialdehyde + NADP+ + H2O | binding structure, overview | Synechococcus sp. ATCC 27264 | succinate + NADPH + 2 H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
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 | Synechococcus sp. |
Synonyms | Comment | Organism |
---|---|---|
SSADH | - |
Synechococcus sp. |
succinic semialdehyde dehydrogenase | - |
Synechococcus sp. |
SySSADH | - |
Synechococcus sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Synechococcus sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.6 | - |
assay at | Synechococcus sp. |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | enzyme activity increases significantly, and the enzyme becomes resistant to oxidative stress in presence of NADP+ and DTT | Synechococcus sp. | |
NADPH | binding structure, overview | Synechococcus sp. |
General Information | Comment | Organism |
---|---|---|
evolution | SSADH belongs to the aldehyde dehydrogenase (ALDH) superfamily | Synechococcus sp. |
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 | Synechococcus sp. |
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 | Synechococcus sp. |