Cloned (Comment) | Organism |
---|---|
gene mdlD, recombinant epression of His-tagged enzyme in Escherichia coli strain BL21(DE3) | Pseudomonas putida |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme in complex with NADP+ and NADP+/benzoate, sitting drop vapour diffusion method, mixing of 10 mg/ml protein in 100 mM HEPES, 100 mM KCl, 2 mM DTT, 2 mM NADP+, pH 7.5, and 1 mM benzoate (for the complex cyrstals), with crystallization solution containing 2.0 M ammonium sulfate and 5% v/v isopropanol, 5-7 days, X-ray diffraction structure determination and analysis at 2.28 A resolution | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
E215D | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Pseudomonas putida |
E215L | site-directed mutagenesis, inactive mutant | Pseudomonas putida |
E215Q | site-directed mutagenesis, inactive mutant | Pseudomonas putida |
E337D | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Pseudomonas putida |
E337L | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Pseudomonas putida |
E337Q | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Pseudomonas putida |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
benzaldehyde + H2O + NADP+ | Pseudomonas putida | - |
benzoate + NADPH + 2 H+ | - |
? | |
benzaldehyde + H2O + NADP+ | Pseudomonas putida ATCC 12633 | - |
benzoate + NADPH + 2 H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | Q84DC3 | - |
- |
Pseudomonas putida ATCC 12633 | Q84DC3 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-chlorobenzaldehyde + H2O + NADP+ | - |
Pseudomonas putida | 4-chlorobenzoate + NADPH + 2 H+ | - |
? | |
4-chlorobenzaldehyde + H2O + NADP+ | - |
Pseudomonas putida ATCC 12633 | 4-chlorobenzoate + NADPH + 2 H+ | - |
? | |
4-methoxybenzaldehyde + H2O + NADP+ | - |
Pseudomonas putida | 4-methoxybenzoate + NADPH + 2 H+ | - |
? | |
4-methoxybenzaldehyde + H2O + NADP+ | - |
Pseudomonas putida ATCC 12633 | 4-methoxybenzoate + NADPH + 2 H+ | - |
? | |
4-nitrobenzaldehyde + H2O + NADP+ | - |
Pseudomonas putida | 4-nitrobenzoate + NADPH + 2 H+ | - |
? | |
4-nitrobenzaldehyde + H2O + NADP+ | - |
Pseudomonas putida ATCC 12633 | 4-nitrobenzoate + NADPH + 2 H+ | - |
? | |
benzaldehyde + H2O + NADP+ | - |
Pseudomonas putida | benzoate + NADPH + 2 H+ | - |
? | |
benzaldehyde + H2O + NADP+ | - |
Pseudomonas putida ATCC 12633 | benzoate + NADPH + 2 H+ | - |
? | |
additional information | specificity data for PpBADH shows that long chain aliphatics containing 5-8 carbons have about twice the relative rate of reactivity than benzaldehyde. As the chain length decreases, the activity declines quickly, with less than 0.1% activity being observed with acetaldehyde. Structure-function analysis | Pseudomonas putida | ? | - |
- |
|
additional information | specificity data for PpBADH shows that long chain aliphatics containing 5-8 carbons have about twice the relative rate of reactivity than benzaldehyde. As the chain length decreases, the activity declines quickly, with less than 0.1% activity being observed with acetaldehyde. Structure-function analysis | Pseudomonas putida ATCC 12633 | ? | - |
- |
Synonyms | Comment | Organism |
---|---|---|
mdlD | - |
Pseudomonas putida |
More | see also EC 1.2.1.28 | Pseudomonas putida |
NAD(P)-dependent benzaldehyde dehydrogenase | - |
Pseudomonas putida |
PpBADH | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
assay at | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | PpBADH is able to utilize both NAD+ and NADP+, selective mechanism, overview | Pseudomonas putida | |
NADP+ | - |
Pseudomonas putida |
General Information | Comment | Organism |
---|---|---|
evolution | benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. The pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs | Pseudomonas putida |
metabolism | benzaldehyde dehydrogenase (PpBADH) is the terminal enzyme in the mandelamide/mandelate pathway of Pseudomonas putida strain ATCC 12633, it catalyzes the conversion of benzaldehyde to benzoic acid with the concomitant reduction of NAD+ (EC 1.2.1.28) or NADP+ (EC 1.2.1.7) to NADH or NADPH, respectively. Benzoic acid subsequently enters the beta-oxoadipate pathway and the citric acid cycle | Pseudomonas putida |
additional information | two conserved glutamates, at positions 215 and 337, act as the general base necessary to hydrolyze the thioacyl intermediate, structure-activity relationship, mechanism, overview. Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs. Structure comparisons | Pseudomonas putida |