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Literature summary extracted from

  • Wang, Y.; Li, P.Y.; Zhang, Y.; Cao, H.Y.; Wang, Y.J.; Li, C.Y.; Wang, P.; Su, H.N.; Chen, Y.; Chen, X.L.; Zhang, Y.Z.
    3,6-Anhydro-L-galactose dehydrogenase VvAHGD is a member of a new aldehyde dehydrogenase family and catalyzes by a novel mechanism with conformational switch of two catalytic residues cysteine 282 and glutamate 248 (2020), J. Mol. Biol., 432, 2186-2203 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.2.1.92 expressed in Escherichia coli BL21(DE3) cells Vibrio variabilis
1.2.1.92 phylogenetic analysis, wild-type and mutant VvAHGDs overexpression in Escherichia coli strain BL21(DE3) Vibrio variabilis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.2.1.92 apo form and in complex with NADP+, hanging drop vapor diffusion method, using 0.2 M NaAC, 0.1 M sodium cacodylate trihydrate (pH 6.5), and 30% (w/v) PEG 8000 Vibrio variabilis
1.2.1.92 purified enzyme in apoform and in complex with NADP+, X-ray diffraction structure determination and analysis at 2.70 A and 2.37 A resolution, respectively, molecular replacement using the structure of Patl_2553 (PDB ID 3K2W) as the starting model Vibrio variabilis

Protein Variants

EC Number Protein Variants Comment Organism
1.2.1.92 C282A inactive Vibrio variabilis
1.2.1.92 E248A inactive Vibrio variabilis
1.2.1.92 E248A site-directed mutagenesis, inactive mutant Vibrio variabilis
1.2.1.92 E383A the mutant shows severely reduced activity compared to the wild type enzyme Vibrio variabilis
1.2.1.92 E443A inactive Vibrio variabilis
1.2.1.92 G206A the mutant shows severely reduced activity compared to the wild type enzyme Vibrio variabilis
1.2.1.92 H449A inactive Vibrio variabilis
1.2.1.92 K173A the mutant shows severely reduced activity compared to the wild type enzyme Vibrio variabilis
1.2.1.92 L249A site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type enzyme Vibrio variabilis
1.2.1.92 L249A the mutant shows slightly reduced activity (about 90%) compared to the wild type enzyme Vibrio variabilis
1.2.1.92 S176A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Vibrio variabilis
1.2.1.92 S176A the mutation dramatically reduces the kcat values of the enzyme against both NADP+ and 3,6-anhydro-alpha-L-galactopyranose and its affinity to NADP+, but has little impact on its affinity to 3,6-anhydro-alpha-L-galactopyranose Vibrio variabilis
1.2.1.92 S227A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Vibrio variabilis
1.2.1.92 S227A the mutation dramatically reduces the kcat values of the enzyme against both NADP+ and 3,6-anhydro-alpha-L-galactopyranose and its affinity to NADP+, but has little impact on its affinity to 3,6-anhydro-alpha-L-galactopyranose Vibrio variabilis
1.2.1.92 S233A site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type enzyme Vibrio variabilis
1.2.1.92 S233A the mutant shows increased activity (about 110%) compared to the wild type enzyme Vibrio variabilis
1.2.1.92 T147A inactive Vibrio variabilis
1.2.1.92 W149A the mutant is almost inactive Vibrio variabilis

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.2.1.92 NaCl about 50% residual activity at 0.5 M, complete inhibition at 2 M Vibrio variabilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.2.1.92 0.33
-
 NADP+ wild type enzyme, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 0.33
-
 NADP+ pH 7.0, 40°C, recombinant wild-type enzyme Vibrio variabilis
1.2.1.92 0.38
-
 NADP+ mutant enzyme S233A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 0.38
-
 NADP+ pH 7.0, 40°C, recombinant mutant S233A Vibrio variabilis
1.2.1.92 0.41
-
 NADP+ mutant enzyme L249A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 0.41
-
 NADP+ pH 7.0, 40°C, recombinant mutant L249A Vibrio variabilis
1.2.1.92 0.58
-
 NADP+ mutant enzyme S227A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 0.58
-
 NADP+ pH 7.0, 40°C, recombinant mutant S227A Vibrio variabilis
1.2.1.92 1.11
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme L249A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 1.11
-
 NADP+ mutant enzyme S176A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 1.11
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant L249A Vibrio variabilis
1.2.1.92 1.11
-
 NADP+ pH 7.0, 40°C, recombinant mutant S176A Vibrio variabilis
1.2.1.92 1.2
-
 3,6-anhydro-alpha-L-galactopyranose wild type enzyme, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 1.2
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant wild-type enzyme Vibrio variabilis
1.2.1.92 1.26
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S227A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 1.26
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S227A Vibrio variabilis
1.2.1.92 1.35
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S233A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 1.35
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S233A Vibrio variabilis
1.2.1.92 1.45
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S176A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 1.45
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S176A Vibrio variabilis

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.2.1.92 intracellular VvAHGD lacks an N-terminal signal peptide sequence based on SignalP 4.1 prediction, consistent with its intracellular location Vibrio variabilis 5622
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.2.1.92 NaCl the intracellular VvAHGD shows weak salt tolerance Vibrio variabilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NAD+ + H2O Vibrio variabilis
-
 3,6-anhydro-L-galactonate + NADH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NAD+ + H2O Vibrio variabilis JCM 19239
-
 3,6-anhydro-L-galactonate + NADH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O Vibrio variabilis
-
 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O Vibrio variabilis JCM 19239
-
 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.2.1.92 Vibrio variabilis
-
-
-
1.2.1.92 Vibrio variabilis A0A090SK43
-
-
1.2.1.92 Vibrio variabilis JCM 19239
-
-
-
1.2.1.92 Vibrio variabilis JCM 19239 A0A090SK43
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.2.1.92 Ni-NTA column chromatography and Superdex 200 gel filtration Vibrio variabilis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NAD(P)+ + H2O = 3,6-anhydro-L-galactonate + NAD(P)H + H+ catalytic mechanism, detailed overview. The catalytic process of VvAHGD involves both the two catalytic residues, Cys282 and Glu248, which not only undergo conformational changes but also function as gatekeepers between the cofactor channel and the substrate channel. The conformational changes of Cys282 and Glu248 lead to the connection and interruption of the cofactor channel and the substrate channel, which promotes the productive binding of NADPþ/L-AHG and the efficient release of NADPH and L-AHGA during the catalysis and therefore leads to the high catalytic activity of VvAHGD Vibrio variabilis
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.2.1.92 57.4
-
 purified recombinant enzyme, substrate 3,6-anhydro-L-galactose (L-AHG), pH 7.0, 40°C Vibrio variabilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NAD+ + H2O
-
 Vibrio variabilis 3,6-anhydro-L-galactonate + NADH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NAD+ + H2O
-
 Vibrio variabilis JCM 19239 3,6-anhydro-L-galactonate + NADH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O
-
 Vibrio variabilis 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O L-AHG is the preferred substrate, and NADP+ is the preferred cofactor Vibrio variabilis 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O
-
 Vibrio variabilis JCM 19239 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
1.2.1.92 3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O L-AHG is the preferred substrate, and NADP+ is the preferred cofactor Vibrio variabilis JCM 19239 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
1.2.1.92 additional information enzyme VvAHGD shows high activity toward 3,6-anhydro-L-galactose (L-AHG). VvAHGD hardly oxidizes lactaldehyde, D-AHG, galactose, or glucose, no or poor activity. Substrate specificity and substrate binding mode in VvAHGD, overview. VvAHGD oxidizes L-AHG using both NADP+ and NAD+ as the cofactor Vibrio variabilis ?
-
-
1.2.1.92 additional information the enzyme hardly oxidizes lactaldehyde, 3,6-anhydro-alpha-D-galactopyranose, D-galactose, or D-glucose Vibrio variabilis ?
-
-
1.2.1.92 additional information the enzyme hardly oxidizes lactaldehyde, 3,6-anhydro-alpha-D-galactopyranose, D-galactose, or D-glucose Vibrio variabilis JCM 19239 ?
-
-
1.2.1.92 additional information enzyme VvAHGD shows high activity toward 3,6-anhydro-L-galactose (L-AHG). VvAHGD hardly oxidizes lactaldehyde, D-AHG, galactose, or glucose, no or poor activity. Substrate specificity and substrate binding mode in VvAHGD, overview. VvAHGD oxidizes L-AHG using both NADP+ and NAD+ as the cofactor Vibrio variabilis JCM 19239 ?
-
-

Subunits

EC Number Subunits Comment Organism
1.2.1.92 ? x * 52600, calculated from amino acid sequence Vibrio variabilis
1.2.1.92 ? x * 52600, about, sequence calculation Vibrio variabilis
1.2.1.92 More AHGD enzyme structure comparisons, overview Vibrio variabilis

Synonyms

EC Number Synonyms Comment Organism
1.2.1.92 3,6-anhydro-L-galactose dehydrogenase
-
 Vibrio variabilis
1.2.1.92 AHGD
-
 Vibrio variabilis
1.2.1.92 VvAHGD
-
 Vibrio variabilis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.2.1.92 40
-
-
 Vibrio variabilis

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.2.1.92 30 45 more than 60% activity between 30 and 45°C Vibrio variabilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.2.1.92 10.67
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S176A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 10.67
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S176A Vibrio variabilis
1.2.1.92 19.15
-
 NADP+ mutant enzyme S176A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 19.15
-
 NADP+ pH 7.0, 40°C, recombinant mutant S176A Vibrio variabilis
1.2.1.92 35.99
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S227A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 35.99
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S227A Vibrio variabilis
1.2.1.92 44.55
-
 NADP+ mutant enzyme S227A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 44.55
-
 NADP+ pH 7.0, 40°C, recombinant mutant S227A Vibrio variabilis
1.2.1.92 52.77
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme L249A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 52.77
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant L249A Vibrio variabilis
1.2.1.92 58.29
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant wild-type enzyme Vibrio variabilis
1.2.1.92 58.29
-
 3,6-anhydro-alpha-L-galactopyranose wild type enzyme, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 63.18
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S233A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 63.18
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S233A Vibrio variabilis
1.2.1.92 63.34
-
 NADP+ mutant enzyme L249A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 63.34
-
 NADP+ pH 7.0, 40°C, recombinant mutant L249A Vibrio variabilis
1.2.1.92 69.09
-
 NADP+ pH 7.0, 40°C, recombinant wild-type enzyme Vibrio variabilis
1.2.1.92 69.09
-
 NADP+ wild type enzyme, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 75.9
-
 NADP+ mutant enzyme S233A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 75.9
-
 NADP+ pH 7.0, 40°C, recombinant mutant S233A Vibrio variabilis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.2.1.92 7
-
-
 Vibrio variabilis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.2.1.92 6.5 8 more than 60% activity at pH 6.5, more than 90% activity from pH 7.0-8.0, 30% activity at pH 9.0 Vibrio variabilis

Cofactor

EC Number Cofactor Comment Organism Structure
1.2.1.92 additional information VvAHGD oxidizes L-AHG using both NADP+ and NAD+ as the cofactor, but VvAHGD prefers NADP+ showing an activity 3.6fold higher compared to NAD+ Vibrio variabilis
1.2.1.92 NAD+
-
 Vibrio variabilis
1.2.1.92 NAD+ the enzyme prefers NADP+, showing an activity 3.6fold higher than that with NAD+ Vibrio variabilis
1.2.1.92 NADP+ NADP+-binding mode in enzyme VvAHGD, overview. One molecule of NADP+ is bound in the active site of each monomer, occupying the cofactor channel. The substrate channel is on the opposite side of the cofactor channel. The catalytic residues Cys282 and Glu248 are located at the junction of these two channels. These two channels are disconnected in the apo structure of VvAHGD, but connected in the structure of the VvAHGD-NADP complex. The cofactor channel has a wide opening on the protein surface, which is mostly positively charged to hold the adenosine monophosphate (AMP) moiety and the diphosphate group of NADP+. The internal part of this channel is negatively charged to accommodate the nicotinamide riboside moiety of NADP+. In the complex, the cofactor NADP+ adopts an extended conformation, which is typical for oxidized NADP+. The VvAHGD-NADP complex, NADP+ is stabilized mainly by hydrogen bond interactions Vibrio variabilis
1.2.1.92 NADP+ the enzyme prefers NADP+, showing an activity 3.6fold higher than that with NAD+ Vibrio variabilis

General Information

EC Number General Information Comment Organism
1.2.1.92 evolution evolutionary relationship between VvAHGD and other ALDHs, a phylogenetic tree and analysis, including VvAHGD and its homologues, lactaldehyde dehydrogenases, succinic semialdehyde dehydrogenases from family ALDH5, betaine aldehyde dehydrogenases from family ALDH25, cytosolic aldehyde dehydrogenases from family ALDH2, and retinaldehyde dehydrogenases from family ALDH1 Vibrio variabilis
1.2.1.92 additional information the catalytic residues of VvAHGD are Cys282 and Glu248 Vibrio variabilis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.2.1.92 7.36
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S176A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 7.36
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S176A Vibrio variabilis
1.2.1.92 17.25
-
 NADP+ mutant enzyme S176A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 17.25
-
 NADP+ pH 7.0, 40°C, recombinant mutant S176A Vibrio variabilis
1.2.1.92 28.56
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S227A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 28.56
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S227A Vibrio variabilis
1.2.1.92 46.8
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme S233A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 46.8
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant S233A Vibrio variabilis
1.2.1.92 47.54
-
 3,6-anhydro-alpha-L-galactopyranose mutant enzyme L249A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 47.54
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant mutant L249A Vibrio variabilis
1.2.1.92 48.575
-
 3,6-anhydro-alpha-L-galactopyranose pH 7.0, 40°C, recombinant wild-type enzyme Vibrio variabilis
1.2.1.92 48.58
-
 3,6-anhydro-alpha-L-galactopyranose wild type enzyme, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 76.81
-
 NADP+ mutant enzyme S227A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 76.81
-
 NADP+ pH 7.0, 40°C, recombinant mutant S227A Vibrio variabilis
1.2.1.92 154.49
-
 NADP+ mutant enzyme L249A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 154.49
-
 NADP+ pH 7.0, 40°C, recombinant mutant L249A Vibrio variabilis
1.2.1.92 199.74
-
 NADP+ mutant enzyme S233A, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 199.74
-
 NADP+ pH 7.0, 40°C, recombinant mutant S233A Vibrio variabilis
1.2.1.92 209.36
-
 NADP+ wild type enzyme, at pH 7.0 and 40°C Vibrio variabilis
1.2.1.92 209.36
-
 NADP+ pH 7.0, 40°C, recombinant wild-type enzyme Vibrio variabilis