Cloned (Comment) | Organism |
---|---|
overexpression of N-terminally His6-tagged HCT in Escherichia coli strain BL21 Star(DE3)pLysS using a commercially available synthetic gene encoding HCT with codons optimized for Escherichia coli expression | Coffea canephora |
Crystallization (Comment) | Organism |
---|---|
purified recombinant HCT, sitting drop technique, mixing of 100 nl of 20 mg/ml protein in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM 2-mercaptoethanol, with 100 nl of precipitant solution containing 1.6 M magnesium sulfate, 0.1 M MES pH 6.5, 20°C, 2 months, X-ray diffraction structure determination and analysis at 3.0 A resolution, molecular replacement | Coffea canephora |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
45000 | 50000 | recombinant HCT, gel filtration | Coffea canephora |
48000 | - |
1 * 48000, recombinant enzyme, SDS-PAGE, 1 * 48464, mass spectrometry | Coffea canephora |
48464 | - |
1 * 48000, recombinant enzyme, SDS-PAGE, 1 * 48464, mass spectrometry | Coffea canephora |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
caffeoyl-CoA + quinate | Coffea canephora | - |
CoA + 5-caffeoylquinate | - |
r | |
caffeoyl-CoA + shikimate | Coffea canephora | - |
CoA + 5-caffeoylshikimate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Coffea canephora | A4ZKE4 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-tagged HCT from Escherichia coli strain BL21 Star(DE3)pLysS by nickel affinity chromatography, dialysis, and cleavage of the tag by TEV protease, followed by gel filtration | Coffea canephora |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
caffeoyl-CoA + quinate | - |
Coffea canephora | CoA + 5-caffeoylquinate | - |
r | |
caffeoyl-CoA + shikimate | - |
Coffea canephora | CoA + 5-caffeoylshikimate | - |
r | |
additional information | HCT has a preference for shikimic acid over quinic acid, substrate specifities of the HCT key enzyme, overview | Coffea canephora | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | 1 * 48000, recombinant enzyme, SDS-PAGE, 1 * 48464, mass spectrometry | Coffea canephora |
More | the enzyme is cleaved into two fragments of 24528 Da and 23906 Da overtime. The protease-cleavage site is localized in the central part of the enzyme, corresponding to a large crossover loop connecting the N- and C-terminal domains in BAHD structures | Coffea canephora |
Synonyms | Comment | Organism |
---|---|---|
HCT | - |
Coffea canephora |
hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase | - |
Coffea canephora |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
additional information | - |
improved stability in sodium phosphate buffer and at higher pH values | Coffea canephora |
General Information | Comment | Organism |
---|---|---|
evolution | HCT belongs to the large plant acyl-CoA-dependent BAHD acyltransferase superfamily | Coffea canephora |
metabolism | the last step in CGA biosynthesis is generally catalysed by a specific hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferase, HQT (c.f. EC 2.3.1.99), but it can also be catalysed by the more widely distributed hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase, HCT, substrate specifities of the HCT key enzyme, overview | Coffea canephora |