Cloned (Comment) | Organism |
---|---|
gene isfD, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) from plasmid HMT-IsfD | Klebsiella oxytoca |
Crystallization (Comment) | Organism |
---|---|
purified His-tagged enzyme IsfD in a ternary complex with NADPH and isethionate, hanging drop vapor diffusion method, mixing of protein in 10 mM HEPES, pH 7.4, 50 mM KCl, 1 mM TCEP (tris(2-carboxyethyl)phosphine), with reservoir solution containing 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 5.5, 30% w/v PEG 4000, 5 mM NADPH, and 0.4 M isethionate, X-ray diffraction structure determination and analysis at 2.8 A resolution, molecular replacement using 2NWQ as a search model, structure comparisons, overview | Klebsiella oxytoca |
Protein Variants | Comment | Organism |
---|---|---|
F191A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
F249A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
I142A | site-directed mutagenesis, the mutant shows 33.6% of wild-type activity | Klebsiella oxytoca |
I186A | site-directed mutagenesis, the mutant shows 25.8% of wild-type activity | Klebsiella oxytoca |
Q244A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
R195A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
R36A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
R37A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
S141A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
T13A | site-directed mutagenesis, the mutant shows 7.8% of wild-type activity | Klebsiella oxytoca |
Y148A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
Y154A | site-directed mutagenesis, inactive mutant | Klebsiella oxytoca |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Klebsiella oxytoca | |
0.0116 | - |
NADP+ | pH 10.0, 22°C, recombinant enzyme | Klebsiella oxytoca | |
50.8 | - |
isethionate | pH 10.0, 22°C, recombinant enzyme | Klebsiella oxytoca |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
101000 | - |
recombinant enzyme, gel filtration | Klebsiella oxytoca |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-sulfoacetaldehyde + NADPH + H+ | Klebsiella oxytoca | - |
isethionate + NADP+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Klebsiella oxytoca | D3U1D9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by streptomycin sulfate precipitation, cobalt affinity chromatography, His-tag cleavage by TEV protease, dialysis, anion exchange chromatography, ultrafiltration, and gel filtration | Klebsiella oxytoca |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-aminoacetaldehyde + CO2 + NADPH + H+ | overall reaction, the enzyme also shows high serine 3-dehydrogenase activity, EC 1.1.1.276 | Klebsiella oxytoca | L-serine + NADP+ | - |
r | |
2-sulfoacetaldehyde + NADPH + H+ | - |
Klebsiella oxytoca | isethionate + NADP+ | - |
r | |
3-hydroxypropionate + NADPH + H+ | - |
Klebsiella oxytoca | ? + NADP+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
homotetramer | 4 * 27000, about, sequence calculation | Klebsiella oxytoca |
More | IsfD forms a homotetramer in both crystal and solution states, with the C-terminal tail of each subunit interacting with the C-terminal tail of the diagonally opposite subunit, forming an antiparallel beta-sheet that constitutes part of the substrate-binding site. The sulfonate group of isethionate is stabilized by a hydrogen bond network formed by the residues Y148, R195, Q244 and a water molecule. In addition, F249 from the diagonal subunit restrains the conformation of Y148 to further stabilize the orientation of the sulfonate group. Quaternary, subunit structure of IsfD and the structure-based sequence alignments of IsfD with selected SDR members, overview | Klebsiella oxytoca |
Synonyms | Comment | Organism |
---|---|---|
IsfD | - |
Klebsiella oxytoca |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | - |
recombinant enzyme | Klebsiella oxytoca |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
58 | - |
melting temperature of enzyme IsfD | Klebsiella oxytoca |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.15 | - |
NADP+ | pH 10.0, 22°C, recombinant enzyme | Klebsiella oxytoca | |
0.15 | - |
isethionate | pH 10.0, 22°C, recombinant enzyme | Klebsiella oxytoca |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
10 | - |
recombinant enzyme | Klebsiella oxytoca |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | no activity with NAD+/NADH | Klebsiella oxytoca | |
NADP+ | - |
Klebsiella oxytoca | |
NADPH | the side chains of T13, R36, R37 form hydrogen bond interactions with the 20-phosphate group of NADPH | Klebsiella oxytoca |
General Information | Comment | Organism |
---|---|---|
evolution | sulfoacetaldehyde reductase (IsfD) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily, involved in nitrogen assimilation from aminoethylsulfonate (taurine) in certain environmental and human commensal bacteria. Biochemical investigations of the substrate scope of IsfD, and bioinformatics analysis of IsfD homologs, suggest that IsfD is related to the promiscuous 3-hydroxyacid dehydrogenases with diverse metabolic functions | Klebsiella oxytoca |
metabolism | IsfD-dependent metabolic pathway and genome neighborhood of IsfD, overview | Klebsiella oxytoca |
additional information | the bound isethionate is oriented with its hydroxyl group facing the tyrosine residue of the catalytic tetrad (Y154) and its sulfonate group forming hydrogen bond network with Y148, R195, Q244 and a water molecule. The side chains of I142, I186 and F191 further stabilize the conformation of the substrate through hydrophobic interactions. Active site structure and structure comparisons, overview | Klebsiella oxytoca |
physiological function | IsfD catalyzes the reversible NADPH-dependent reduction of sulfoacetaldehyde, which is generated by transamination of taurine, forming hydroxyethylsulfonate (isethionate) as a waste product | Klebsiella oxytoca |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.003 | - |
isethionate | pH 10.0, 22°C, recombinant enzyme | Klebsiella oxytoca | |
12.93 | - |
NADP+ | pH 10.0, 22°C, recombinant enzyme | Klebsiella oxytoca |