Literature summary for 1.2.1.16 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.

Activating Compound

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

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli strain BL21(DE3)pLysS	Pseudomonas putida
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

Cloned (Comment)

Organism

expressed in Escherichia coli strain BL21(DE3)pLysS

Pseudomonas putida

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

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

General Stability

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

KM Value [mM]

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

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
45000	-	2 * 45000, in solution, SDS-PAGE,	Pseudomonas putida
47400	-	calculated from amino acid sequence	Pseudomonas putida
90000	-	SDS-PAGE	Pseudomonas putida
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)

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

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	-	strain ATCC 12633	-

Purification (Commentary)

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

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
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

Storage Stability	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3-chlorobenzaldehyde + NAD+ + H2O	-	Pseudomonas putida	3-chlorobenzoate + NADH + 2 H+	-	?
3-hydroxy-benzaldehyde + NAD+ + H2O	good substrate	Pseudomonas putida	3-hydroxybenzoate + NADH + H+	-	?
3-hydroxybenzaldehyde + NAD+ + H2O	-	Pseudomonas putida	3-hydroxybenzoate + NADH + 2 H+	-	?
4-chloro-benzaldehyde + NAD+ + H2O	good substrate	Pseudomonas putida	4-chlorobenzoate + NADH + H+	-	?
4-chlorobenzaldehyde + NAD+ + H2O	-	Pseudomonas putida	4-chlorobenzoate + NADH + 2 H+	-	?
benzaldehyde + NAD+ + H2O	-	Pseudomonas putida	benzoate + NADH + H+	-	?
benzaldehyde + NAD+ + H2O	mandelate pathway	Pseudomonas putida	benzoate + NADH + H+	-	ir
benzaldehyde + NADP+ + H2O	weak reaction	Pseudomonas putida	benzoate + NADPH + H+	-	?
benzaldehyde + NADP+ + H2O	mandelate pathway	Pseudomonas putida	benzoate + NADPH + 2 H+	-	ir
cyclohexanal + NAD+ + H2O	good substrate	Pseudomonas putida	cyclohexanecarboxylic acid + NADH + H+	-	?
cyclohexanal + NAD+ + H2O	-	Pseudomonas putida	? + NADH + 2 H+	-	?
heptanal + NAD+ + H2O	good substrate	Pseudomonas putida	heptanoate + NADH + H+	-	?
hexanal + NAD+ + H2O	good substrate	Pseudomonas putida	hexanoate + NADH + H+	-	?
hexanal + NAD+ + H2O	-	Pseudomonas putida	hexanoate + NADH + 2 H+	-	?
additional information	no activity with 2-chlorobenzaldehyde, 3-chlorobenzaldehyde, and acetaldehyde	Pseudomonas putida	?	-	?
additional information	no activity is observed with 2-chlorobenzaldehyde	Pseudomonas putida	?	-	?
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	?	-	?
octanal + NAD+ + H2O	good substrate	Pseudomonas putida	octanoate + NADH + H+	-	?
pentanal + NAD+ + H2O	good substrate	Pseudomonas putida	pentanoate + NADH + H+	-	?
pentanal + NAD+ + H2O	-	Pseudomonas putida	pentanoate + NADH + 2 H+	-	?
phenylacetaldehyde + NAD+ + H2O	-	Pseudomonas putida	phenylacetate + NADH + H+	-	?

Subunits

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

Synonyms

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

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
17	-	NADP+	native wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C	Pseudomonas putida
17	-	NADP+	wild-type BADH	Pseudomonas putida
21	-	NADP+	His-tagged BADH	Pseudomonas putida
21.1	-	NADP+	recombinant wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C	Pseudomonas putida
56	-	NAD+	wild-type BADH	Pseudomonas putida
56.8	-	NAD+	native wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C	Pseudomonas putida
66	-	NAD+	recombinant wild type enzyme, in TAPS buffer, at pH 8.5, at 30°C	Pseudomonas putida
66	-	NAD+	His-tagged BADH	Pseudomonas putida

pH Optimum

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

pH Range

pH Minimum	pH Maximum	Comment	Organism
additional information	-	The pH profiles are bell-shaped indicating that two ionizable groups are involved in the catalytic mechanism.	Pseudomonas putida
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

pH Stability	pH Stability Maximum	Comment	Organism
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

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