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Literature summary extracted from

  • Gmelch, T.J.; Sperl, J.M.; Sieber, V.
    Molecular dynamics analysis of a rationally designed aldehyde dehydrogenase gives insights into improved activity for the non-native cofactor NAD (2020), ACS Synth. Biol., 9, 920-929 .
    View publication on PubMed

Application

EC Number Application Comment Organism
1.2.1.4 synthesis the aldehyde dehydrogenase from Thermoplasma acidophilum implemented as a key enzyme in a synthetic cellfree reaction cascade for the production of alcohols. Thermoplasma acidophilum enzyme TaALDH matches the cascade equirements regarding temperature stability, efficient heterologous expression in Escherichia coli, and exclusive activity for D-glyceraldehyde (not active on acetaldehyde). The remaining drawback is its cofactor preference toward NADP+ resulting in high KM for NAD+ and a rather low overall activity under cascade conditions Thermoplasma acidophilum

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.4 D176S site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 D176S/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 D176S/S206K site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 D176S/S206K/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 D176S/S206K/M262I/W271Y/W275V site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 F34M/Y399C/S405N site-directed mutagenesis, crystal structure analysis Thermoplasma acidophilum
1.2.1.4 M262I site-directed mutagenesis, the mutation results in 30% higher activity with NAD+ compared to wild-type enzyme, the surface mutation M262I has influence on the solubility Thermoplasma acidophilum
1.2.1.4 additional information random mutagenesis approach for the aldehyde dehydrogenase of Thermoplasma acidophilum (TaALDH) to increase volumetric activity and slightly improve NAD+ acceptance. Roughly 450 mutants do not show any activity, and around 400 mutants have an activity below the template (M33) threshold. Saturation mutagenesis of the residues at the entrance of the substrate pocket can eliminate substrate inhibition. Molecular dynamics simulations show a significant gain of flexibility at the cofactor binding site for the final variant Thermoplasma acidophilum
1.2.1.4 S175E site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/D176S site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/D176S/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/D176S/S206K site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/D176S/S206K/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/S206K site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S175E/S206K/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S206K site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum
1.2.1.4 S206K/M262I site-directed mutagenesis, the mutation results in increased activity with NAD+ compared to NADP+ and the wild-type enzyme Thermoplasma acidophilum

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.2.1.4 D-glyceraldehyde substrate inhibition Thermoplasma acidophilum

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.4 Thermoplasma acidophilum Q9HL01
-
-
1.2.1.4 Thermoplasma acidophilum AMRC-C165 Q9HL01
-
-
1.2.1.4 Thermoplasma acidophilum ATCC 25905 Q9HL01
-
-
1.2.1.4 Thermoplasma acidophilum JCM 9062 Q9HL01
-
-
1.2.1.4 Thermoplasma acidophilum NBRC 15155 Q9HL01
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.4 D-glyceraldehyde + NADP+
-
 Thermoplasma acidophilum D-glycerate + NADPH + H+
-
 ?
1.2.1.4 D-glyceraldehyde + NADP+
-
 Thermoplasma acidophilum JCM 9062 D-glycerate + NADPH + H+
-
 ?
1.2.1.4 D-glyceraldehyde + NADP+
-
 Thermoplasma acidophilum AMRC-C165 D-glycerate + NADPH + H+
-
 ?
1.2.1.4 D-glyceraldehyde + NADP+
-
 Thermoplasma acidophilum ATCC 25905 D-glycerate + NADPH + H+
-
 ?
1.2.1.4 D-glyceraldehyde + NADP+
-
 Thermoplasma acidophilum NBRC 15155 D-glycerate + NADPH + H+
-
 ?
1.2.1.4 additional information no activity with acetaldehyde Thermoplasma acidophilum ?
-
-
1.2.1.4 additional information no activity with acetaldehyde Thermoplasma acidophilum JCM 9062 ?
-
-
1.2.1.4 additional information no activity with acetaldehyde Thermoplasma acidophilum AMRC-C165 ?
-
-
1.2.1.4 additional information no activity with acetaldehyde Thermoplasma acidophilum ATCC 25905 ?
-
-
1.2.1.4 additional information no activity with acetaldehyde Thermoplasma acidophilum NBRC 15155 ?
-
-

Synonyms

EC Number Synonyms Comment Organism
1.2.1.4 ALDH
-
 Thermoplasma acidophilum
1.2.1.4 Ta0439
-
 Thermoplasma acidophilum
1.2.1.4 TaAlDH
-
 Thermoplasma acidophilum

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.2.1.4 7 9.5 Tm of wild-type enzyme Thermoplasma acidophilum
1.2.1.4 72.5
-
 Tm of mutant D176S/S206K/M262I/W271Y/W275V Thermoplasma acidophilum
1.2.1.4 78.5
-
 Tm of mutant D176S/S206K/M262I Thermoplasma acidophilum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.4 7.3
-
 assay at Thermoplasma acidophilum

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.4 additional information increased to preferred activity of enzyme mutants with NAD+ compared to NADP+ in contrast to wild-type enzyme Thermoplasma acidophilum
1.2.1.4 NADP+ the wild-type enzyme strongly prefers NADP+, negligible activity with NAD+ Thermoplasma acidophilum

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.2.1.4 1.25
-
 D-glyceraldehyde pH 7.3, temperature not specified in the publication, wild-type enzyme Thermoplasma acidophilum
1.2.1.4 3.8
-
 D-glyceraldehyde pH 7.3, temperature not specified in the publication, mutant D176S/S206K/M262I Thermoplasma acidophilum
1.2.1.4 31.2
-
 D-glyceraldehyde pH 7.3, temperature not specified in the publication, mutant D176S/S206K/M262I/W271Y/W275V Thermoplasma acidophilum

General Information

EC Number General Information Comment Organism
1.2.1.4 additional information structural modeling and docking, the three-dimensional structure of wild-type TaALDH is modeled using the available crystal structure of the triple mutant TaALDH F34M/Y399C/S405N (PDB ID 5M4X) as a template, molecular dynamics simulations Thermoplasma acidophilum