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 (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 | 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 | Comment | Organism | Structure |
---|---|---|---|
NaCl | about 50% residual activity at 0.5 M, complete inhibition at 2 M | Vibrio variabilis |
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 | 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 | Comment | Organism | Structure |
---|---|---|---|
NaCl | the intracellular VvAHGD shows weak salt tolerance | Vibrio variabilis |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Vibrio variabilis | - |
- |
- |
Vibrio variabilis | A0A090SK43 | - |
- |
Vibrio variabilis JCM 19239 | - |
- |
- |
Vibrio variabilis JCM 19239 | A0A090SK43 | - |
- |
Purification (Comment) | Organism |
---|---|
Ni-NTA column chromatography and Superdex 200 gel filtration | Vibrio variabilis |
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 |
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 | 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 | 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 | Comment | Organism |
---|---|---|
3,6-anhydro-L-galactose dehydrogenase | - |
Vibrio variabilis |
AHGD | - |
Vibrio variabilis |
VvAHGD | - |
Vibrio variabilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
- |
Vibrio variabilis |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | 45 | more than 60% activity between 30 and 45°C | Vibrio variabilis |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
- |
Vibrio variabilis |
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 | 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 | 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 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 |