NAD-specific glutamate dehydrogenase A from Halobacterium salinarum

Enzyme Description

Species
Halobacterium salinarum β†—
Extremophile
Yes "halophilic" organism
EC Number
1.4.1.2 β†—

Sequence

Length: 434 amino acids
TMASKSDSTHDESGDEAADSTEPESALETARRQLYHAASYLDIDQNIVERLKYPKKVHEVTIPIERDDGTVEVFTGYRAQHDSVRGPYKGGLRYHPDVTRDECVGLGMWMTWKCAVMDLPFGGAKGGVAVNPKELSPEEKERLTRRFTQEIRDVIGPNQDIPAPDMGTDPQTMAWLMDAYSMQEGETTPGVVTGKPPVVGGSEGREEAPGRSVAIITQLVCEYYDQPLDETTVAVQGYGSVGANAARLLDKWGATIVAISDVNGAMYEPDGIDTASVPSHDEEPEAVTTYADTVISNEELLTLDVDVLIPAALGNVITKENAEAIAADLVVEGANGPTTSTADSILADRDVAVIPDILANAGGVTVSYFEWLQDINRRAWSLERVNDELEAEMQAAWRAVKDEYENRDVTWRDAAYIVALSRIAEAHEARGLWP
Nayla Munawar et al. (2012) β€” Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase
Extremophiles  Β· doi:10.1007/s00792-012-0491-7 β†—  Β· Activity - Classical Stability - Incubation
18 measurements ▼
Database ID
UniProt: P29051 β†—
Sequence Annotation
Inferred (first residue from UniProt sequence removed from mature protein, 3 mutations)
Protein Source
Recombinant, host archaeon Haloferax volcanii DS70

Experimental Data (18 measurements)

18 measurements
Mutation Property Assay Solvent Solvent Volume Aqueous ReferenceWT Reference Measured Value Units Solution pH Temperature Substrate(s) Product(s) Cofactor(s) Shaking Comments
K89L/A163G/S367A Activity - Classical Activity measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in the presence of organic solvent Dimethyl Sulfoxide (DMSO) 10% 100.0 β€” 105.0 % 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA 10 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Classical aqueous control (in %) | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Activity - Classical Activity measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in the presence of organic solvent Methanol 10% 100.0 β€” 38.0 % 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA 10 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Classical aqueous control (in %) | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Activity - Classical Activity measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in the presence of organic solvent Ethanol 10% 100.0 β€” 23.0 % 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA 10 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Classical aqueous control (in %) | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Activity - Classical Activity measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in the presence of organic solvent Acetonitrile 10% 100.0 β€” 5.4 % 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA 10 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Classical aqueous control (in %) | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (24 hours at 70Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethyl Sulfoxide (DMSO) 20% 100.0 β€” 96.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: 70Β°C, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (0 hour at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Methanol 10% 100.0 β€” 104.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (24 hours at 70Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Acetonitrile 20% 100.0 β€” 38.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: 70Β°C, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (24 hours at 70Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Ethanol 20% 100.0 β€” 36.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: 70Β°C, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (24 hours at 70Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Methanol 20% 100.0 β€” 108.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: 70Β°C, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (24 hours at 70Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Ethanol 10% 100.0 β€” 0.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: 70Β°C, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (24 hours at 70Β°C), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Methanol 10% 100.0 β€” 78.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: 70Β°C, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (8 days at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethyl Sulfoxide (DMSO) 10% 100.0 β€” 100.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (8 days at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Acetonitrile 10% 100.0 β€” 100.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (8 days at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Ethanol 10% 100.0 β€” 122.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (8 days at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Methanol 10% 100.0 β€” 104.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (0 hour at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Dimethyl Sulfoxide (DMSO) 10% 100.0 β€” 100.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (0 hour at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Acetonitrile 10% 100.0 β€” 106.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate
K89L/A163G/S367A Stability - Incubation Activity after incubation (0 hour at room temperature), measured by absorbance spectrophotometry (NADH absorbance measurement, 340 nm) in aqueous phase Ethanol 10% 100.0 β€” 121.0 % Incubation: ?, Assay: 80 mM glycine–NaOH, 0.9 M NaCl, 2.6 mM EDTA Incubation: ?, Assay: 10 Incubation: Room temperature, Assay: 40Β°C 100 mM L-Methionine or L-Norleucine NH3​ , NH3 or 2-oxohexanoate , Ξ±-keto-Ξ³-methiolbutyrate 3 mM NAD+ β€” Water-incubated control (in %), assay in aqueous phase | Mutant enzyme does not accept L-glutamate as substrate, unlike the WT, unclear substrate

Mutations in this dataset (1)

K89L/A163G/S367A

Visualization : Activity β€” Classical

One bar per measurement. Colour = solvent, shade = solvent volume.

Visualization : Stability β€” Incubation

One bar per measurement. Colour = solvent, shade = solvent volume.

Structure

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