Cloned (Comment) | Organism |
---|---|
five putative SDH-encoding genes amplified and cloned into either a modified pET28a vector or the p15TVLIC vector, a ligation-independent vector due to incompatible cloning sites between the insert and pET28.3. Expressed in either the Escherichia coli strain BL21 gold (p15TV-LIC constructs) or the Escherichia coli BL21CodonPlus (pET28.3constructs) | Pseudomonas putida KT2440 |
SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into a vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics. | Pseudomonas putida |
SDH-encoding gene is amplified by polymerase chain reaction from Pseudomonas putida KT2440 genomic DNA using primers with incorporated restriction sites. Resulting product is cloned into vector and expressed in the Escherichia coli strain BL21 with appropriate antibiotics. | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | no measurable activity with NAD+ | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with NADH+ | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with quinate | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with shikimate | Pseudomonas putida | |
0.0046 | - |
shikimate | pH 8.8, presence of 2 mM NADP+ | Pseudomonas putida | |
0.0073 | - |
NADP+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
0.0786 | - |
shikimate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
0.0786 | - |
shikimate | in the presence of 2 mM NAD+ | Pseudomonas putida KT2440 | |
0.105 | - |
shikimate | pH 8.8, presence of 2 mM NADP+ | Pseudomonas putida | |
0.105 | - |
shikimate | in the presence of 2 mM NADP+ | Pseudomonas putida KT2440 | |
0.112 | - |
NADP+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
0.112 | - |
NADP+ | in the presence of shikimate | Pseudomonas putida KT2440 | |
0.351 | - |
shikimate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
0.572 | - |
NAD+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
0.783 | - |
quinate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
1.083 | - |
NAD+ | pH 8.8, presence of 2 mM quinate | Pseudomonas putida | |
4.2 | - |
shikimate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
5.4 | - |
NAD+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
5.43 | - |
NAD+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
5.43 | - |
NAD+ | in the presence of shikimate | Pseudomonas putida KT2440 |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
quinate + NAD+ | Pseudomonas putida | the fourth enzyme in the shikimate pathway | 3-dehydroquinate + NADH + H+ | - |
r | |
quinate + NAD+ | Pseudomonas putida KT 2240 | the fourth enzyme in the shikimate pathway | 3-dehydroquinate + NADH + H+ | - |
r | |
shikimate + NAD+ | Pseudomonas putida | - |
3-dehydroshikimate + NADH + H+ | - |
r | |
shikimate + NAD+ | Pseudomonas putida KT 2240 | - |
3-dehydroshikimate + NADH + H+ | - |
r | |
shikimate + NADP+ | Pseudomonas putida | the fourth enzyme in the shikimate pathway | 3-dehydroshikimate + NADPH + H+ | - |
r | |
shikimate + NADP+ | Pseudomonas putida KT 2240 | the fourth enzyme in the shikimate pathway | 3-dehydroshikimate + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | Q88GF6 | homolog sdhL | - |
Pseudomonas putida | Q88IJ7 | homolog ael1 | - |
Pseudomonas putida | Q88JP1 | homolog rifl | - |
Pseudomonas putida | Q88K85 | homolog ydiB | - |
Pseudomonas putida | Q88RQ5 | homolog aroE | - |
Pseudomonas putida KT 2240 | Q88GF6 | homolog sdhL | - |
Pseudomonas putida KT 2240 | Q88IJ7 | homolog ael1 | - |
Pseudomonas putida KT 2240 | Q88JP1 | homolog rifl | - |
Pseudomonas putida KT 2240 | Q88K85 | homolog ydiB | - |
Pseudomonas putida KT 2240 | Q88RQ5 | homolog aroE | - |
Pseudomonas putida KT2440 | Q88IJ7 | - |
- |
Pseudomonas putida KT2440 | Q88RQ5 | - |
- |
Purification (Comment) | Organism |
---|---|
by nickel-nitrilotriacetic acid affinity chromatography | Pseudomonas putida KT2440 |
Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4 °C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol. | Pseudomonas putida |
Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4°C in 10 mM Tris-HCl, 500 mM NaCl, and 5% glycerol. | Pseudomonas putida |
Cultures with expressed SDH enzymes are incubated and then induced with 0.4 mM isopropyl-beta-D-thiogalactopyranoside. Harvested cells are disrupted and the insoluble cellular material is removed by centrifugation. The recombinants are purified from other contaminating proteins using nickel-nitrilotriacetic acid affinity chromatography. Protein samples for kinetic studies are dialyzed and stored at 4°C in 10 mM TrisHCl, 500 mM NaCl, and 5% glycerol. | Pseudomonas putida |
Storage Stability | Organism |
---|---|
4°C, 10 mM Tris-HCl buffer, pH 7.5, 500 mM NaCl, 5% glycerol | Pseudomonas putida KT2440 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | broad extent to which the SDH enzyme superfamily has diversified. 5 evolutionarily distinct SDH homologs in the genome of the common soil-inhabiting bacterium, Pseudomonas putida KT2440 | Pseudomonas putida KT2440 | ? | - |
? | |
quinate + NAD(P)+ | - |
Pseudomonas putida KT2440 | 3-dehydroquinate + NAD(P)H + H+ | - |
? | |
quinate + NAD+ | the fourth enzyme in the shikimate pathway | Pseudomonas putida | 3-dehydroquinate + NADH + H+ | - |
r | |
quinate + NAD+ | the fourth enzyme in the shikimate pathway | Pseudomonas putida KT 2240 | 3-dehydroquinate + NADH + H+ | - |
r | |
shikimate + NAD(P)+ | - |
Pseudomonas putida KT2440 | 3-dehydroshikimate + NAD(P)H + H+ | - |
? | |
shikimate + NAD+ | - |
Pseudomonas putida | 3-dehydroshikimate + NADH + H+ | - |
r | |
shikimate + NAD+ | - |
Pseudomonas putida KT2440 | 3-dehydroshikimate + NADH + H+ | - |
r | |
shikimate + NAD+ | - |
Pseudomonas putida KT 2240 | 3-dehydroshikimate + NADH + H+ | - |
r | |
shikimate + NADP+ | the fourth enzyme in the shikimate pathway | Pseudomonas putida | 3-dehydroshikimate + NADPH + H+ | - |
r | |
shikimate + NADP+ | the fourth enzyme in the shikimate pathway | Pseudomonas putida KT 2240 | 3-dehydroshikimate + NADPH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
ael1 | - |
Pseudomonas putida |
ael1 | - |
Pseudomonas putida KT2440 |
AroE | - |
Pseudomonas putida |
AroE | - |
Pseudomonas putida KT2440 |
rifI | - |
Pseudomonas putida |
SDH | - |
Pseudomonas putida |
SDH | - |
Pseudomonas putida KT2440 |
sdhL | - |
Pseudomonas putida |
shikimate dehydrogenase | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at | Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | no measurable activity with NAD+ | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with NADH | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with NADH+ | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with quinate | Pseudomonas putida | |
additional information | - |
additional information | no measurable activity with shikimate | Pseudomonas putida | |
0.077 | - |
NADP+ | in the presence of quinate | Pseudomonas putida KT2440 | |
0.16 | - |
quinate | in the presence of 2 mM NADP+ | Pseudomonas putida KT2440 | |
1.99 | - |
shikimate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
1.99 | - |
shikimate | in the presence of 2 mM NAD+ | Pseudomonas putida KT2440 | |
5.3 | - |
NADP+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
5.3 | - |
NADP+ | in the presence of shikimate | Pseudomonas putida KT2440 | |
5.8 | - |
shikimate | pH 8.8, presence of 2 mM NADP+ | Pseudomonas putida | |
5.8 | - |
shikimate | in the presence of 2 mM NADP+ | Pseudomonas putida KT2440 | |
6.72 | - |
NAD+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
6.72 | - |
NAD+ | in the presence of shikimate | Pseudomonas putida KT2440 | |
31.2 | - |
shikimate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
31.2 | - |
shikimate | in the presence of 2 mM NAD+ | Pseudomonas putida KT2440 | |
37.7 | - |
quinate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
40.7 | - |
NAD+ | pH 8.8, presence of 2 mM quinate | Pseudomonas putida | |
55.7 | - |
shikimate | pH 8.8, presence of 2 mM NAD+ | Pseudomonas putida | |
61.1 | - |
NAD+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
97.1 | - |
NAD+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
97.1 | - |
NAD+ | in the presence of shikimate | Pseudomonas putida KT2440 | |
261.5 | - |
NADP+ | pH 8.8, presence of 2 mM shikimate | Pseudomonas putida | |
261.5 | - |
NADP+ | in the presence of shikimate | Pseudomonas putida KT2440 | |
266.2 | - |
shikimate | pH 8.8, presence of 2 mM NADP+ | Pseudomonas putida | |
266.2 | - |
shikimate | in the presence of 2 mM NADP+ | Pseudomonas putida KT2440 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.8 | - |
assay at | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD(P)+ | - |
Pseudomonas putida KT2440 | |
NAD+ | - |
Pseudomonas putida | |
NADP+ | - |
Pseudomonas putida |