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Literature summary for 1.1.1.313 extracted from

  • Zhou, Y.; Wei, Y.; Lin, L.; Xu, T.; Ang, E.L.; Zhao, H.; Yuchi, Z.; Zhang, Y.
    Biochemical and structural investigation of sulfoacetaldehyde reductase from Klebsiella oxytoca (2019), Biochem. J., 476, 733-746 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene isfD, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) from plasmid HMT-IsfD Klebsiella oxytoca

Crystallization (Commentary)

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

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 [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 [Da] Molecular Weight Maximum [Da] Comment Organism
101000
-
recombinant enzyme, gel filtration Klebsiella oxytoca

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-sulfoacetaldehyde + NADPH + H+ Klebsiella oxytoca
-
isethionate + NADP+
-
r

Organism

Organism UniProt Comment Textmining
Klebsiella oxytoca D3U1D9
-
-

Purification (Commentary)

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 and Products (Substrate)

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

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

Synonyms Comment Organism
IsfD
-
Klebsiella oxytoca

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
recombinant enzyme Klebsiella oxytoca

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
58
-
melting temperature of enzyme IsfD Klebsiella oxytoca

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
10
-
recombinant enzyme Klebsiella oxytoca

Cofactor

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

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 [mM/s]

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