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Literature summary extracted from

  • Zahniser, M.P.D.; Prasad, S.; Kneen, M.M.; Kreinbring, C.A.; Petsko, G.A.; Ringe, D.; McLeish, M.J.
    Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the Class 3 aldehyde dehydrogenase superfamily (2017), Protein Eng. Des. Sel., 30, 271-278 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.7 gene mdlD, recombinant epression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Pseudomonas putida
1.2.1.28 gene mdlD, recombinant epression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.1.7 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

EC Number Protein Variants Comment Organism
1.2.1.7 E215D site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.7 E215L site-directed mutagenesis, inactive mutant Pseudomonas putida
1.2.1.7 E215Q site-directed mutagenesis, inactive mutant Pseudomonas putida
1.2.1.7 E337D site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.7 E337L site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.7 E337Q site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.28 E215D site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.28 E215L site-directed mutagenesis, inactive mutant Pseudomonas putida
1.2.1.28 E215Q site-directed mutagenesis, inactive mutant Pseudomonas putida
1.2.1.28 E337D site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.28 E337L site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida
1.2.1.28 E337Q site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.28 0.0012
-
 4-Chlorobenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 0.002
-
 4-methoxybenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 0.0044
-
 benzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 0.0066
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337D, with NAD+ Pseudomonas putida
1.2.1.28 0.0093
-
 4-nitrobenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 0.02
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337L, with NAD+ Pseudomonas putida
1.2.1.28 0.02
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337Q, with NAD+ Pseudomonas putida
1.2.1.28 0.028
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E215D, with NAD+ Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.1.7 benzaldehyde + H2O + NADP+ Pseudomonas putida
-
 benzoate + NADPH + 2 H+
-
 ?
1.2.1.7 benzaldehyde + H2O + NADP+ Pseudomonas putida ATCC 12633
-
 benzoate + NADPH + 2 H+
-
 ?
1.2.1.28 benzaldehyde + H2O + NAD+ Pseudomonas putida
-
 benzoate + NADH + 2 H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.7 Pseudomonas putida Q84DC3
-
-
1.2.1.7 Pseudomonas putida ATCC 12633 Q84DC3
-
-
1.2.1.28 Pseudomonas putida Q84DC3
-
-
1.2.1.28 Pseudomonas putida ATCC 12633 Q84DC3
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.7 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Pseudomonas putida
1.2.1.28 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.7 4-chlorobenzaldehyde + H2O + NADP+
-
 Pseudomonas putida 4-chlorobenzoate + NADPH + 2 H+
-
 ?
1.2.1.7 4-chlorobenzaldehyde + H2O + NADP+
-
 Pseudomonas putida ATCC 12633 4-chlorobenzoate + NADPH + 2 H+
-
 ?
1.2.1.7 4-methoxybenzaldehyde + H2O + NADP+
-
 Pseudomonas putida 4-methoxybenzoate + NADPH + 2 H+
-
 ?
1.2.1.7 4-methoxybenzaldehyde + H2O + NADP+
-
 Pseudomonas putida ATCC 12633 4-methoxybenzoate + NADPH + 2 H+
-
 ?
1.2.1.7 4-nitrobenzaldehyde + H2O + NADP+
-
 Pseudomonas putida 4-nitrobenzoate + NADPH + 2 H+
-
 ?
1.2.1.7 4-nitrobenzaldehyde + H2O + NADP+
-
 Pseudomonas putida ATCC 12633 4-nitrobenzoate + NADPH + 2 H+
-
 ?
1.2.1.7 benzaldehyde + H2O + NADP+
-
 Pseudomonas putida benzoate + NADPH + 2 H+
-
 ?
1.2.1.7 benzaldehyde + H2O + NADP+
-
 Pseudomonas putida ATCC 12633 benzoate + NADPH + 2 H+
-
 ?
1.2.1.7 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 ?
-
-
1.2.1.7 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 ?
-
-
1.2.1.28 4-chlorobenzaldehyde + H2O + NADP+
-
 Pseudomonas putida 4-chlorobenzoate + NADPH + 2 H+
-
 ?
1.2.1.28 4-methoxybenzaldehyde + H2O + NADP+
-
 Pseudomonas putida 4-methoxybenzoate + NADPH + 2 H+
-
 ?
1.2.1.28 4-nitrobenzaldehyde + H2O + NADP+
-
 Pseudomonas putida 4-nitrobenzoate + NADPH + 2 H+
-
 ?
1.2.1.28 benzaldehyde + H2O + NAD+
-
 Pseudomonas putida benzoate + NADH + 2 H+
-
 ?
1.2.1.28 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 ?
-
-

Synonyms

EC Number Synonyms Comment Organism
1.2.1.7 mdlD
-
 Pseudomonas putida
1.2.1.7 More see also EC 1.2.1.28 Pseudomonas putida
1.2.1.7 NAD(P)-dependent benzaldehyde dehydrogenase
-
 Pseudomonas putida
1.2.1.7 PpBADH
-
 Pseudomonas putida
1.2.1.28 mdlD
-
 Pseudomonas putida
1.2.1.28 More see also EC 1.2.1.7 Pseudomonas putida
1.2.1.28 NAD(P)-dependent benzaldehyde dehydrogenase
-
 Pseudomonas putida
1.2.1.28 PpBADH
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.1.7 30
-
 assay at Pseudomonas putida
1.2.1.28 30
-
 assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.1.28 0.8
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337Q, with NAD+ Pseudomonas putida
1.2.1.28 1.3
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337L, with NAD+ Pseudomonas putida
1.2.1.28 1.5
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E215D, with NAD+ Pseudomonas putida
1.2.1.28 45
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337D, with NAD+ Pseudomonas putida
1.2.1.28 95
-
 4-nitrobenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 116
-
 4-methoxybenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 135
-
 4-Chlorobenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 156
-
 benzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.7 8.5
-
 assay at Pseudomonas putida
1.2.1.28 8.5
-
 assay at Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.7 additional information PpBADH is able to utilize both NAD+ and NADP+, selective mechanism, overview Pseudomonas putida
1.2.1.7 NADP+
-
 Pseudomonas putida
1.2.1.28 additional information PpBADH is able to utilize both NAD+ and NADP+, selective mechanism, overview Pseudomonas putida
1.2.1.28 NAD+
-
 Pseudomonas putida
1.2.1.28 NADP+
-
 Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.2.1.7 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
1.2.1.7 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
1.2.1.7 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
1.2.1.28 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
1.2.1.28 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
1.2.1.28 additional information two conserved glutamates, at positions 215 and 337, might 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

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.2.1.28 40
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337Q, with NAD+ Pseudomonas putida
1.2.1.28 53.6
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E215D, with NAD+ Pseudomonas putida
1.2.1.28 65
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337L, with NAD+ Pseudomonas putida
1.2.1.28 6818.2
-
 benzaldehyde pH 8.5, 30°C, recombinant mutant E337D, with NAD+ Pseudomonas putida
1.2.1.28 10215
-
 4-nitrobenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 35454
-
 benzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 58000
-
 4-methoxybenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida
1.2.1.28 112500
-
 4-Chlorobenzaldehyde pH 8.5, 30°C, recombinant wild-type enzyme, with NAD+ Pseudomonas putida