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Literature summary extracted from

  • Crabo, A.G.; Singh, B.; Nguyen, T.; Emami, S.; Gassner, G.T.; Sazinsky, M.H.
    Structure and biochemistry of phenylacetaldehyde dehydrogenase from the Pseudomonas putida S12 styrene catabolic pathway (2017), Arch. Biochem. Biophys., 616, 47-58 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.39 gene styD, DNA and amino acid sequence determination and analysis, recombinant expression of N-terminally His-tagged enzyme, NPADH, in Escherichia coli strain BL21(DE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.1.39 purified recombinant His-tagged enzyme, sitting drop vapor diffusion method, mixing of 5-7 mg/ml protein in 10 mM Tris, pH 7.5, and 1 mM 2-mercaptoethanol, with 100-200 mM trisodium citrate, pH 8.5, and 12-16% PEG 3350, at 4°C, X-ray diffraction structure determination and analysis at 2.83 A resolution, molecular replacement using ALDH1, PDB ID 1BXS, as a starting model, model building Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.2.1.39 Mg2+ inhibits and activates Pseudomonas putida
1.2.1.39 Mn2+ inhibits and activates Pseudomonas putida
1.2.1.39 NAD+ substrate inhibition, competitive binding of NADH Pseudomonas putida
1.2.1.39 NADH product inhibition, competitive binding of NAD+. For many aldehyde dehydrogenases, NADH binds competitively with NAD+ and forms a nonproductive dead-end complex during catalysis. In the absence of styrene monooxygenase reductase, which regenerates NAD+ from NADH in the first step of styrene catabolism, NPADH is inhibited by a ternary complex involving NADH, product, and phenylacetaldehyde, substrate Pseudomonas putida
1.2.1.39 PMSF inactivates NPADH, presumably by modifying the active site cysteine Pseudomonas putida
1.2.1.39 Pyridine nucleotides titrations of NPADH with NADþ and NADH are evaluated to estimate the binding affinities of the oxidized and reduced pyridine nucleotides under equilibrium conditions. Mg2+ is included in these studies Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.39 additional information
-
 additional information kinetic analysis Pseudomonas putida
1.2.1.39 0.0487
-
 NAD+ pH 8.5, 25°C Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.2.1.39 Mg2+ the enzyme includes both an activating and inhibitory metal binding site in the catalytic mechanism of NPADH. The activating divalent metal binding site may be best described as the direct interaction of the metal ion with the pyrophosphate linkage joining the nicotinamide mononucleotide and adenosine mononucleotide components of the pyridine nucleotide structure. A second mononuclear metal binding site, occupied by Mg2+ is detected in this structure. The Mg2+ in this site assumes a roughly octahedral geometry and is coordinated by the backbone carbonyl oxygens of Val40, Asp109, Glu196, and Val345, as well as a monodentate interaction with a carboxylate oxygen of Asp109. The sixth ligand is a crystallographically resolved water molecule Pseudomonas putida
1.2.1.39 Mn2+ inhibits and activates Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.2.1.39 227000
-
 recombinant His-tagged enzyme, gel filtration Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.1.39 phenylacetaldehyde + NAD+ + H2O Pseudomonas putida
-
 phenylacetate + NADH + 2 H+
-
 ?
1.2.1.39 phenylacetaldehyde + NAD+ + H2O Pseudomonas putida S12
-
 phenylacetate + NADH + 2 H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.39 Pseudomonas putida V4GH04
-
-
1.2.1.39 Pseudomonas putida S12 V4GH04
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.39 recombinant N-terminally His-tagged enzyme, NPADH, from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration to over 90% purity Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.2.1.39 phenylacetaldehyde + NAD+ + H2O = phenylacetate + NADH + H+ sequential reaction mechanism in which NAD+ serves as both the leading substrate and homotropic allosteric activator, catalytic mechanism involving E169, E267, and C301, overview. The catalytic Glu has two conformations: a passive conformer that tucks away to allow hydride transfer to the nicotinamide ring, and an active conformer that abstracts a proton from the thioester-deacylating water Pseudomonas putida
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.39 phenylacetaldehyde + NAD+ + H2O
-
 Pseudomonas putida phenylacetate + NADH + 2 H+
-
 ?
1.2.1.39 phenylacetaldehyde + NAD+ + H2O
-
 Pseudomonas putida S12 phenylacetate + NADH + 2 H+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.2.1.39 homotetramer 4 * 55000, recombinant His-tagged enzyme, SDS-PAGE Pseudomonas putida
1.2.1.39 More the oligomerization domains of all four subunits form the core of PADH and are responsible not only for the effective dimerization observed within the homotetramer, but also for the association of these dimers to form the tetramer. Each 496 amino acid PADH subunit consists of three domains: an N-terminal NADþ-binding domain (residues 1-130 and 159-269), a catalytic domain (residues 270-471), and an oligomerization domain (131-158 and 472-496). The enzyme has a unique set of intersubunit interactions and active site tunnel for substrate entrance. Each oligomerization domain of NPADH contains a six-residue insertion that extends this loop over the substrate entrance tunnel of a neighboring subunit, thereby obstructing the active site of the adjacent subunit. This feature might be an important factor in the homotropic activation and product inhibition mechanisms. The substrate channel of NPADH is narrower and lined with more aromatic residues, which include Phe170, Phe295, Phe466, and Trp177, suggesting a means for enhancing substrate specificity Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.2.1.39 NPADH
-
 Pseudomonas putida
1.2.1.39 PADH
-
 Pseudomonas putida
1.2.1.39 phenylacetaldehyde dehydrogenase
-
 Pseudomonas putida
1.2.1.39 styD
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.1.39 25
-
 assay at Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.39 8 10
-
 Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.39 NAD+ N-terminal NAD+-binding domain, comprising residues 1-130 and 159-269, structure analysis, overview Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.2.1.39 metabolism the enzyme catalyzes a step in the styrene catabolic pathway of Pseudomonas putida Pseudomonas putida
1.2.1.39 additional information substrate channel and active site structure, overview. A majority of conserved residues in NPADH localize to the active site and NAD+-binding pocket. At the interface between the two pockets are the catalytic Cys 301 and Glu 267 residues, which serve as the general nucleophile and general base for the reaction, respectively Pseudomonas putida
1.2.1.39 physiological function phenylacetaldehyde dehydrogenase catalyzes the NAD+-dependent oxidation of phenylactealdehyde to phenylacetic acid in the styrene catabolic and detoxification pathway of Pseudomonas putida strain S12 Pseudomonas putida