Cloned (Comment) | Organism |
---|---|
genes of wild-type and its truncational mutant proteins (TM1, TM2, TM3, and TM4) amplified and cloned into the pGEM-T vector and subcloned into a modified pET-28a expression vector. Truncated mutant protein TM5 amplified and annealed to the pET-46 Ek/LIC vector. Expressed in Escherichia coli BL21(DE3) CodonPlus RIPL-competent cells | Fibrobacter succinogenes subsp. succinogenes S85 |
Protein Variants | Comment | Organism |
---|---|---|
D270A | displays catalytic activity against tetraacetyl-xylopyranoside | Fibrobacter succinogenes subsp. succinogenes S85 |
D270N | displays catalytic activity against tetraacetyl-xylopyranoside | Fibrobacter succinogenes subsp. succinogenes S85 |
E194A | displays some catalytic activity | Fibrobacter succinogenes subsp. succinogenes S85 |
E194A/D270A | activity is completely abolished | Fibrobacter succinogenes subsp. succinogenes S85 |
E194N | exhibits detectable catalytic activity | Fibrobacter succinogenes subsp. succinogenes S85 |
H273Q | activity is completely abolished | Fibrobacter succinogenes subsp. succinogenes S85 |
additional information | truncational mutant proteins TM1 (esterase domain plus carbohydrate-binding module), TM2 (esterase domain), TM3 (carbohydrate-binding module plus FPm-1 domain), TM4 (carbohydrate-binding module) and TM5 (FPm-1 domain). Truncation of FPm-1 leads to a dramatic reduction of binding activity for the TM1 mutant. TM1 and TM2 have no discernible affinity for insoluble oat spelt xylan, whereas TM3, TM4, and TM5 bind to this substrate | Fibrobacter succinogenes subsp. succinogenes S85 |
S44G | activity is completely abolished | Fibrobacter succinogenes subsp. succinogenes S85 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.07 | - |
tetraacetyl-xylopyranoside | mutant TM2, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
0.08 | - |
tetraacetyl-xylopyranoside | wild-type, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
0.09 | - |
tetraacetyl-xylopyranoside | mutant TM1, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
0.2 | - |
tetraacetyl-xylopyranoside | mutants E194A, D270N and D270A, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
7 | - |
tetraacetyl-xylopyranoside | mutant E194N, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Fibrobacter succinogenes subsp. succinogenes S85 | Q9F106 | - |
- |
Purification (Comment) | Organism |
---|---|
truncational mutant proteins purified to near homogeneity | Fibrobacter succinogenes subsp. succinogenes S85 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
insoluble oat spelt xylan | - |
Fibrobacter succinogenes subsp. succinogenes S85 | ? | - |
? | |
additional information | wild-type protein does not show any binding activity to Avicel. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function (Fibrobacter succinogenes-specific paralogous module 1, FPm-1). The N-terminal half of Axe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan is determined to originate from FPm-1. Esterase activity is derived from a tetrad composed of Ser44, His273, Glu194, and Asp270, with both Glu194 and Asp270 functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis | Fibrobacter succinogenes subsp. succinogenes S85 | ? | - |
? | |
tetraacetyl-xylopyranoside + H2O | - |
Fibrobacter succinogenes subsp. succinogenes S85 | ? + acetate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
acetyl xylan esterase | - |
Fibrobacter succinogenes subsp. succinogenes S85 |
Axe6B | - |
Fibrobacter succinogenes subsp. succinogenes S85 |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.8 | - |
tetraacetyl-xylopyranoside | mutant D270A, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
2 | - |
tetraacetyl-xylopyranoside | mutant D270N, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
2.8 | - |
tetraacetyl-xylopyranoside | mutant E194N, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
2.9 | - |
tetraacetyl-xylopyranoside | mutant E194A, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
13 | - |
tetraacetyl-xylopyranoside | mutant TM2, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
15 | - |
tetraacetyl-xylopyranoside | wild-type and mutant TM1, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Fibrobacter succinogenes subsp. succinogenes S85 | mutant TM4, calculated from sequence | - |
4.6 |
Fibrobacter succinogenes subsp. succinogenes S85 | mutant TM2, calculated from sequence | - |
5.2 |
Fibrobacter succinogenes subsp. succinogenes S85 | mutant TM5, calculated from sequence | - |
10.1 |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.4 | - |
tetraacetyl-xylopyranoside | mutant E194N, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
9 | - |
tetraacetyl-xylopyranoside | mutant D270A, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
10 | - |
tetraacetyl-xylopyranoside | mutant D270N, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
14 | - |
tetraacetyl-xylopyranoside | mutant E194A, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
170 | - |
tetraacetyl-xylopyranoside | mutant TM1, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 | |
190 | - |
tetraacetyl-xylopyranoside | wild-type and mutant TM2, at 37°C, in 100 mM Tris-HCl buffer, pH 7.4, and 3 mM MgCl2 | Fibrobacter succinogenes subsp. succinogenes S85 |