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Literature summary for 1.2.1.92 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
Search for more literature for 1.2.1.92

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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
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

Protein Variants Comment Organism
C282A inactive Vibrio variabilis
E248A inactive Vibrio variabilis
E248A site-directed mutagenesis, inactive mutant Vibrio variabilis
E383A the mutant shows severely reduced activity compared to the wild type enzyme Vibrio variabilis
E443A inactive Vibrio variabilis
G206A the mutant shows severely reduced activity compared to the wild type enzyme Vibrio variabilis
H449A inactive Vibrio variabilis
K173A the mutant shows severely reduced activity compared to the wild type enzyme Vibrio variabilis
L249A site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type enzyme Vibrio variabilis
L249A the mutant shows slightly reduced activity (about 90%) compared to the wild type enzyme Vibrio variabilis
S176A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Vibrio variabilis
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
S227A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Vibrio variabilis
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
S233A site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type enzyme Vibrio variabilis
S233A the mutant shows increased activity (about 110%) compared to the wild type enzyme Vibrio variabilis
T147A inactive Vibrio variabilis
W149A the mutant is almost inactive Vibrio variabilis

Inhibitors

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

KM Value [mM]

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

Localization

Localization Comment Organism GeneOntology No. Textmining
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

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Vibrio variabilis
-
-
-
Vibrio variabilis A0A090SK43
-
-
Vibrio variabilis JCM 19239
-
-
-
Vibrio variabilis JCM 19239 A0A090SK43
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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]

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3,6-anhydro-alpha-L-galactopyranose + NAD+ + H2O
-
 Vibrio variabilis 3,6-anhydro-L-galactonate + NADH + H+
-
 ?
3,6-anhydro-alpha-L-galactopyranose + NAD+ + H2O
-
 Vibrio variabilis JCM 19239 3,6-anhydro-L-galactonate + NADH + H+
-
 ?
3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O
-
 Vibrio variabilis 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
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+
-
 ?
3,6-anhydro-alpha-L-galactopyranose + NADP+ + H2O
-
 Vibrio variabilis JCM 19239 3,6-anhydro-L-galactonate + NADPH + H+
-
 ?
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+
-
 ?
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 ?
-
-
additional information the enzyme hardly oxidizes lactaldehyde, 3,6-anhydro-alpha-D-galactopyranose, D-galactose, or D-glucose Vibrio variabilis ?
-
-
additional information the enzyme hardly oxidizes lactaldehyde, 3,6-anhydro-alpha-D-galactopyranose, D-galactose, or D-glucose Vibrio variabilis JCM 19239 ?
-
-
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

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

Synonyms

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

Temperature Optimum [°C]

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

Temperature Range [°C]

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

Turnover Number [1/s]

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

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
-
 Vibrio variabilis

pH Range

pH Minimum pH Maximum Comment Organism
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

Cofactor Comment Organism Structure
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
NAD+
-
 Vibrio variabilis
NAD+ the enzyme prefers NADP+, showing an activity 3.6fold higher than that with NAD+ Vibrio variabilis
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
NADP+ the enzyme prefers NADP+, showing an activity 3.6fold higher than that with NAD+ Vibrio variabilis

General Information

General Information Comment Organism
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
additional information the catalytic residues of VvAHGD are Cys282 and Glu248 Vibrio variabilis

kcat/KM [mM/s]

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