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Literature summary 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)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.313 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

EC Number Protein Variants Comment Organism
1.1.1.313 F191A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 F249A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 I142A site-directed mutagenesis, the mutant shows 33.6% of wild-type activity Klebsiella oxytoca
1.1.1.313 I186A site-directed mutagenesis, the mutant shows 25.8% of wild-type activity Klebsiella oxytoca
1.1.1.313 Q244A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 R195A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 R36A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 R37A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 S141A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 T13A site-directed mutagenesis, the mutant shows 7.8% of wild-type activity Klebsiella oxytoca
1.1.1.313 Y148A site-directed mutagenesis, inactive mutant Klebsiella oxytoca
1.1.1.313 Y154A site-directed mutagenesis, inactive mutant Klebsiella oxytoca

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.313 additional information
-
 additional information Michaelis-Menten kinetics Klebsiella oxytoca
1.1.1.313 0.0116
-
 NADP+ pH 10.0, 22°C, recombinant enzyme Klebsiella oxytoca
1.1.1.313 50.8
-
 isethionate pH 10.0, 22°C, recombinant enzyme Klebsiella oxytoca

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.313 101000
-
 recombinant enzyme, gel filtration Klebsiella oxytoca

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.313 2-sulfoacetaldehyde + NADPH + H+ Klebsiella oxytoca
-
 isethionate + NADP+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.313 Klebsiella oxytoca D3U1D9
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.313 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.313 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
1.1.1.313 2-sulfoacetaldehyde + NADPH + H+
-
 Klebsiella oxytoca isethionate + NADP+
-
 r
1.1.1.313 3-hydroxypropionate + NADPH + H+
-
 Klebsiella oxytoca ? + NADP+
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.1.313 homotetramer 4 * 27000, about, sequence calculation Klebsiella oxytoca
1.1.1.313 additional information 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

EC Number Synonyms Comment Organism
1.1.1.313 IsfD
-
 Klebsiella oxytoca

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.313 40
-
 recombinant enzyme Klebsiella oxytoca

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.313 58
-
 melting temperature of enzyme IsfD Klebsiella oxytoca

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.313 0.15
-
 isethionate pH 10.0, 22°C, recombinant enzyme Klebsiella oxytoca
1.1.1.313 0.15
-
 NADP+ pH 10.0, 22°C, recombinant enzyme Klebsiella oxytoca

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.313 10
-
 recombinant enzyme Klebsiella oxytoca

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.313 additional information no activity with NAD+/NADH Klebsiella oxytoca
1.1.1.313 NADP+
-
 Klebsiella oxytoca
1.1.1.313 NADPH the side chains of T13, R36, R37 form hydrogen bond interactions with the 20-phosphate group of NADPH Klebsiella oxytoca

General Information

EC Number General Information Comment Organism
1.1.1.313 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
1.1.1.313 metabolism IsfD-dependent metabolic pathway and genome neighborhood of IsfD, overview Klebsiella oxytoca
1.1.1.313 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
1.1.1.313 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.313 0.003
-
 isethionate pH 10.0, 22°C, recombinant enzyme Klebsiella oxytoca
1.1.1.313 12.93
-
 NADP+ pH 10.0, 22°C, recombinant enzyme Klebsiella oxytoca