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Literature summary extracted from

  • Yeung, C.K.; Yep, A.; Kenyon, G.L.; McLeish, M.J.
    Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633 (2008), Biochim. Biophys. Acta, 1784, 1248-1255.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.2.1.16 dithiothreitol necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme Pseudomonas putida

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.16 expressed in Escherichia coli strain BL21(DE3)pLysS Pseudomonas putida
1.2.1.16 Protein expression in Escherichia coli strain JM109. Single colonies are picked and the DNA is isolated and screened for the desired mutation either by restriction analysis (C103A and C249A) or by sequencing (C140A and C220A). The overall fidelity of the PCR amplification and the presence of the desired mutations are confirmed by sequencing. The plasmids containing the mutated genes are then transformed into Escherichia coli strain BL21(DE3)pLysS for protein expression. Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.16 C102A mutant exhibits BADH activity between 50 and 75% that of the wild type enzyme Pseudomonas putida
1.2.1.16 C103A enzyme is inactive Pseudomonas putida
1.2.1.16 C140A mutant exhibits BADH activity between 50 and 75% that of the wild type enzyme Pseudomonas putida
1.2.1.16 C140A activity between 50 and 75% in comparison to wild-type Pseudomonas putida
1.2.1.16 C220A mutant exhibits BADH activity between 50 and 75% that of the wild type enzyme Pseudomonas putida
1.2.1.16 C220A activity between 50 and 75% in comparison to wild-type Pseudomonas putida
1.2.1.16 C249A inactive Pseudomonas putida
1.2.1.16 C249A activity between 50 and 75% in comparison to wild-type Pseudomonas putida

General Stability

EC Number General Stability Organism
1.2.1.16 dithiothreitol is necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.16 0.011
-
 benzaldehyde recombinant wild type enzyme, in the presence of NADP+, TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.011
-
 benzaldehyde His-tagged BADH, cosubstrate NADP+ Pseudomonas putida
1.2.1.16 0.013
-
 benzaldehyde native wild type enzyme, in the presence of NADP+, TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.013
-
 benzaldehyde wild-type BADH, cosubstrate NADP+ Pseudomonas putida
1.2.1.16 0.033
-
 benzaldehyde native wild type enzyme, in the presence of NAD+, TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.033
-
 benzaldehyde wild-type BADH, cosubstrate NAD+ Pseudomonas putida
1.2.1.16 0.035
-
 benzaldehyde recombinant wild type enzyme, in the presence of NAD+, TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.035
-
 benzaldehyde His-tagged BADH, cosubstrate NAD+ Pseudomonas putida
1.2.1.16 0.111
-
 NAD+ recombinant wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.111
-
 NAD+ His-tagged BADH, cosubstrate benzaldehyde Pseudomonas putida
1.2.1.16 0.134
-
 NAD+ native wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.134
-
 NAD+ wild-type BADH, cosubstrate benzaldehyde Pseudomonas putida
1.2.1.16 0.22
-
 NADP+ native wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.22
-
 NADP+ wild-type BADH, cosubstrate benzaldehyde Pseudomonas putida
1.2.1.16 0.287
-
 NADP+ recombinant wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 0.287
-
 NADP+ His-tagged BADH, cosubstrate benzaldehyde Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.2.1.16 45000
-
 2 * 45000, in solution, SDS-PAGE, Pseudomonas putida
1.2.1.16 47400
-
 calculated from amino acid sequence Pseudomonas putida
1.2.1.16 90000
-
 SDS-PAGE Pseudomonas putida
1.2.1.16 90000
-
 Dimer is determined by gel filtration whereas the molecular mass of the enzyme is calculated by comparison to the relative mobility of the standard proteins. Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.1.16 benzaldehyde + NAD+ + H2O Pseudomonas putida mandelate pathway benzoate + NADH + H+
-
 ir
1.2.1.16 benzaldehyde + NADP+ + H2O Pseudomonas putida mandelate pathway benzoate + NADPH + 2 H+
-
 ir

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.16 Pseudomonas putida
-
 strain ATCC 12633
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.16 Ni2+-agarose chromatography Pseudomonas putida
1.2.1.16 Purification of his-tagged proteins by using a standard Ni2+-agarose chromatography protocol. Pseudomonas putida

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.2.1.16 additional information
-
 Good activity with several alternate substrates including the aromatic substrates, 4-chloro- and 3-hydroxy-benzaldehyde, as well as cyclohexanal. BADH is most active with medium chain aliphatic substrates such as pentanal and hexanal.The level of activity with aliphatic substrates drops off rapidly as the chain length decreased with very little activity (<0.1%) being observed with acetaldehyde. Pseudomonas putida

Storage Stability

EC Number Storage Stability Organism
1.2.1.16 Addition of 2 mM DTT to both stacking and resolving gels results in a decrease in the protein laddering effect, and both BADH and BADH-His are present as near-homogeneous species. Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.16 3-chlorobenzaldehyde + NAD+ + H2O
-
 Pseudomonas putida 3-chlorobenzoate + NADH + 2 H+
-
 ?
1.2.1.16 3-hydroxy-benzaldehyde + NAD+ + H2O good substrate Pseudomonas putida 3-hydroxybenzoate + NADH + H+
-
 ?
1.2.1.16 3-hydroxybenzaldehyde + NAD+ + H2O
-
 Pseudomonas putida 3-hydroxybenzoate + NADH + 2 H+
-
 ?
1.2.1.16 4-chloro-benzaldehyde + NAD+ + H2O good substrate Pseudomonas putida 4-chlorobenzoate + NADH + H+
-
 ?
1.2.1.16 4-chlorobenzaldehyde + NAD+ + H2O
-
 Pseudomonas putida 4-chlorobenzoate + NADH + 2 H+
-
 ?
1.2.1.16 benzaldehyde + NAD+ + H2O
-
 Pseudomonas putida benzoate + NADH + H+
-
 ?
1.2.1.16 benzaldehyde + NAD+ + H2O mandelate pathway Pseudomonas putida benzoate + NADH + H+
-
 ir
1.2.1.16 benzaldehyde + NADP+ + H2O weak reaction Pseudomonas putida benzoate + NADPH + H+
-
 ?
1.2.1.16 benzaldehyde + NADP+ + H2O mandelate pathway Pseudomonas putida benzoate + NADPH + 2 H+
-
 ir
1.2.1.16 cyclohexanal + NAD+ + H2O good substrate Pseudomonas putida cyclohexanecarboxylic acid + NADH + H+
-
 ?
1.2.1.16 cyclohexanal + NAD+ + H2O
-
 Pseudomonas putida ? + NADH + 2 H+
-
 ?
1.2.1.16 heptanal + NAD+ + H2O good substrate Pseudomonas putida heptanoate + NADH + H+
-
 ?
1.2.1.16 hexanal + NAD+ + H2O good substrate Pseudomonas putida hexanoate + NADH + H+
-
 ?
1.2.1.16 hexanal + NAD+ + H2O
-
 Pseudomonas putida hexanoate + NADH + 2 H+
-
 ?
1.2.1.16 additional information no activity with 2-chlorobenzaldehyde, 3-chlorobenzaldehyde, and acetaldehyde Pseudomonas putida ?
-
 ?
1.2.1.16 additional information no activity is observed with 2-chlorobenzaldehyde Pseudomonas putida ?
-
 ?
1.2.1.16 additional information the 3- and 4-chloro substituents as well as the 3-hydroxy substituent are all electron withdrawing. All these compounds show a reduction in reaction velocity from that of the unsubstituted benzaldehyde Pseudomonas putida ?
-
 ?
1.2.1.16 octanal + NAD+ + H2O good substrate Pseudomonas putida octanoate + NADH + H+
-
 ?
1.2.1.16 pentanal + NAD+ + H2O good substrate Pseudomonas putida pentanoate + NADH + H+
-
 ?
1.2.1.16 pentanal + NAD+ + H2O
-
 Pseudomonas putida pentanoate + NADH + 2 H+
-
 ?
1.2.1.16 phenylacetaldehyde + NAD+ + H2O
-
 Pseudomonas putida phenylacetate + NADH + H+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.2.1.16 dimer in solution Pseudomonas putida
1.2.1.16 dimer 2 * 45000, in solution, SDS-PAGE, Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.2.1.16 BADH
-
 Pseudomonas putida
1.2.1.16 NAD(P)+-dependent benzaldehyde dehydrogenase
-
 Pseudomonas putida
1.2.1.16 NAD(P)-dependent benzaldehyde dehydrogenase
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.1.16 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
1.2.1.16 17
-
 NADP+ native wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 17
-
 NADP+ wild-type BADH Pseudomonas putida
1.2.1.16 21
-
 NADP+ His-tagged BADH Pseudomonas putida
1.2.1.16 21.1
-
 NADP+ recombinant wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 56
-
 NAD+ wild-type BADH Pseudomonas putida
1.2.1.16 56.8
-
 NAD+ native wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 66
-
 NAD+ recombinant wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C Pseudomonas putida
1.2.1.16 66
-
 NAD+ His-tagged BADH Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.16 8.5
-
 assay at Pseudomonas putida

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.2.1.16 additional information
-
 The pH profiles are bell-shaped indicating that two ionizable groups are involved in the catalytic mechanism. Pseudomonas putida
1.2.1.16 5 10 KM value for NAD+ increases from 0.3 to 1 mM as the pH changes from pH 5 to 10. The pH profile data are obtained at a NAD+ concentration of 5 mM, essentially saturating across the entire pH range. Similarly, data for the NADP+ pH profile are obtained at 10 mM NADP+ Pseudomonas putida

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.2.1.16 5 10 the Km value for NAD+ increases from 0.3 to 1 mM as the pH changes from pH 5 to 10 Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.16 NAD+
-
 Pseudomonas putida
1.2.1.16 NAD+ preferred cofactor Pseudomonas putida
1.2.1.16 NADP+
-
 Pseudomonas putida
1.2.1.16 NADP+ less active with NADP+ compared to NAD+ Pseudomonas putida