Cloned (Comment) | Organism |
---|---|
recombinant enzyme expression as N-terminally His-tagged, functional dimeric type and mutant enzyme proteins lacking the mitochondrial localization sequence | Glycine max |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type and K95A mutant enzymes, hanging drops vapor diffusion method , 0004 ml of 10-12 mg/ml protein solution is mixed with an equal volume of crystallization solution containing 10% PEG 8000, 0.2 M NaCl, and 0.1 sodium/potassium phosphate, pH 7.0, and 0.2 M MgSO4, at 4°C over a 0.7 mL reservoir. The K95A mutant is crystallized in similar drops from a solution containing 20% PEG 3350, 0.2 M potassium citrate, pH 7.5, X-ray diffraction structure determination and analysis at 2.50 A and 1.77 A resolution, respectively, molecular replacement | Glycine max |
Protein Variants | Comment | Organism |
---|---|---|
K95A | site-directed mutagenesis, inactive mutant | Glycine max |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.26 | - |
hydrogen cyanide | recombinant enzyme, pH 9.0, 25°C | Glycine max | |
0.81 | - |
L-cysteine | recombinant enzyme, pH 9.0, 25°C | Glycine max |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
mitochondrion | the enzyme has a mitochondrial localization sequence | Glycine max | 5739 | - |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-cysteine + hydrogen cyanide | Glycine max | - |
hydrogen sulfide + L-3-cyanoalanine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Glycine max | I1L6I6 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant functional N-terminally His-tagged wild-type and mutant enzymes lacking the mitochondrial localization sequence by nickel affinity chromatography and gel filtration | Glycine max |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-cysteine + hydrogen cyanide | - |
Glycine max | hydrogen sulfide + L-3-cyanoalanine | - |
? | |
L-cysteine + hydrogen cyanide | the enzyme forms an alpha-aminoacrylate reaction intermediate from L-Cys | Glycine max | hydrogen sulfide + L-3-cyanoalanine | - |
? | |
additional information | the enzyme displays a 230fold higher catalytic efficiency with Cys than O-acetylserine, cf. EC 2.5.1.47 | Glycine max | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | The N-terminal domain (residues 94 to 197) is formed by a central beta-sheet (beta3 to beta5) surrounded by four alpha-helices (alpha1 to alpha4). In between the two domains, alpha5 (residues 195 to 2019) spans both halves of the structure. The C-terminal domain consists of a short beta-sheet (beta1 to beta2, residues 51 to 93) and a larger beta-sheet (beta6 to beta8) surrounded by three alpha-helices (alpha6 to alpha8). An additional alpha-helix (alpha9) forms part of the dimer interface by packing against alpha2 and alpha3 of the adjoining monomer | Glycine max |
Synonyms | Comment | Organism |
---|---|---|
CAS | - |
Glycine max |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Glycine max |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
38.9 | - |
L-cysteine | recombinant enzyme, pH 9.0, 25°C | Glycine max | |
39.2 | - |
hydrogen cyanide | recombinant enzyme, pH 9.0, 25°C | Glycine max |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
9 | - |
assay at | Glycine max |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | binding site determination, each monomer consists of two mixed alpha/eta domains with a pyridoxal 5'-phosphate cofactor at the active site separating the two halves | Glycine max |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme is a member of the beta-substituted alanine synthase (BSAS) family, structure and substrate specificity comparisons with another family member, O-acetylserine sulfhydrylase, EC 2.5.1.47, from Glycine max. This enzyme prefers O-acetylserine versus Cys by 23fold and sulfide by 270fold over CN- | Glycine max |
additional information | comparison of the open active site wild-type enzyme and the closed active site K95A mutant monomer structures, overview | Glycine max |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
48.02 | - |
L-cysteine | recombinant enzyme, pH 9.0, 25°C | Glycine max | |
150.8 | - |
hydrogen cyanide | recombinant enzyme, pH 9.0, 25°C | Glycine max |