Protein Variants | Comment | Organism |
---|---|---|
A155H | drastic increases in Km values | Escherichia coli |
A155W | 13fold increase in Km value for dihydropteroate | Escherichia coli |
D151A | mutation in residue specific for dihydropteroate binding, mutant is defective in using tetrahydrofolate as substrate | Lacticaseibacillus casei |
D154A | mutation in residue specific for dihydropteroate binding, 200fold increase in Km value for substrate 5,6,7,8-tetrahydropteroyl-gamma-(L-Glu)2 | Escherichia coli |
F121A | 50fold increase in Km for 5,10-methylene-5,6,7,8-tetrahydrofolic acid | Lacticaseibacillus casei |
F75A | increase in Km value for 5,10-methylene-5,6,7,8-tetrahydrofolic acid, decreases in Km values for ATP and L-Glu | Lacticaseibacillus casei |
additional information | construction of domain-swap chimera proteins between the Escherichia coli and the Lactobacillus casei enzymes. Chimera possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Mutants with swapped omega loops, containing a folate binding site, retain the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating Lactobacillus casei folypolyglutamate synthetase to contain an Escherichia coli folypolyglutamate synthetase dihydropteroate binding loop does not alter its substrate specificity to using dihydropteroate as a substrate | Lacticaseibacillus casei |
additional information | expression of the amino-terminal domain with residues 1-287. The domain binds tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. Construction of domain-swap chimera proteins between the Escherichia coli and the Lactobacillus casei enzymes. Chimera possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Mutants with swapped omega loops, containing a folate binding site, retain the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating Lactobacillus casei folypolyglutamate synthetase to contain an Escherichia coli folypolyglutamate synthetase dihydropteroate binding loop does not alter its substrate specificity to using dihydropteroate as a substrate | Escherichia coli |
R15E | increases in Km values | Lacticaseibacillus casei |
S152A | decrease in Vmax | Lacticaseibacillus casei |
S152W | increases in Km values | Lacticaseibacillus casei |
T119W | 100fold increase in Km for 5,10-methylene-5,6,7,8-tetrahydrofolic acid | Lacticaseibacillus casei |
T122H | drastic increases in Km values | Escherichia coli |
T122W | drastic increases in Km values | Escherichia coli |
Y414A | decrease in Km values for ATP, increase in Km for L-Glu | Lacticaseibacillus casei |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0063 | - |
7,8-dihydropteroate | wild-type, pH 9.8 | Lacticaseibacillus casei | |
0.0063 | - |
7,8-dihydropteroate | wild-type, pH 9.8 | Escherichia coli | |
0.01 | - |
5,6,7,8-tetrahydrofolic acid | mutant A155W, pH 9.8 | Escherichia coli | |
0.01 | - |
5,10-methylene-5,6,7,8-tetrahydrofolyl-(L-Glu)2 | mutant D151A, pH 9.8 | Lacticaseibacillus casei | |
0.032 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | wild-type | Lacticaseibacillus casei | |
0.034 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant S152A, pH 9.8 | Lacticaseibacillus casei | |
0.037 | - |
ATP | mutant D154A, pH 9.8 cosubstrate 7,8-dihydropteroate | Escherichia coli | |
0.048 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant Y414A, pH 9.8 | Lacticaseibacillus casei | |
0.05 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | wild-type | Escherichia coli | |
0.053 | - |
L-Glu | mutant F75A, pH 9.8 | Lacticaseibacillus casei | |
0.054 | - |
ATP | wild-type, pH 9.8 | Escherichia coli | |
0.065 | - |
ATP | mutant A155H, pH 9.8 cosubstrate 7,8-dihydropteroate | Escherichia coli | |
0.071 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, pH 9.8 | Lacticaseibacillus casei | |
0.071 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, pH 9.8 | Escherichia coli | |
0.076 | - |
ATP | mutant A155H, pH 9.8, cosubstrate 5,6,7,8-tetrahydrofolic acid | Escherichia coli | |
0.08 | - |
7,8-dihydropteroate | mutant A155W, pH 9.8 | Escherichia coli | |
0.084 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant R15E, pH 9.8 | Lacticaseibacillus casei | |
0.11 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant D151A, pH 9.8 | Lacticaseibacillus casei | |
0.116 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant S152W, pH 9.8 | Lacticaseibacillus casei | |
0.118 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | chimera containing N-terminal residues 1-71 of Lactobacillus casei enzyme, residues 82-93 of Escherichia coli enzyme, and residues 83-428 of Lactobacillus casei enzyme, pH 9.8 | Lacticaseibacillus casei | |
0.118 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | chimera containing N-terminal residues 1-71 of Lactobacillus casei enzyme, residues 82-93 of Escherichia coli enzyme, and residues 83-428 of Lactobacillus casei enzyme, pH 9.8 | Escherichia coli | |
0.12 | - |
L-Glu | mutant A155H, pH 9.8, cosubstrate 5,6,7,8-tetrahydrofolic acid | Escherichia coli | |
0.134 | - |
7,8-dihydropteroate | chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, pH 9.8 | Lacticaseibacillus casei | |
0.134 | - |
7,8-dihydropteroate | chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, pH 9.8 | Escherichia coli | |
0.14 | - |
7,8-dihydropteroate | mutant D154A, pH 9.8 | Escherichia coli | |
0.15 | - |
7,8-dihydropteroate | mutant T122W, pH 9.8 | Escherichia coli | |
0.17 | - |
L-Glu | mutant A155H, pH 9.8, cosubstrate 7,8-dihydropteroate | Escherichia coli | |
0.18 | - |
ATP | mutant F75A, pH 9.8 | Lacticaseibacillus casei | |
0.2 | - |
L-Glu | mutant D154A, pH 9.8, cosubstrate 5,6,7,8-tetrahydrofolic acid | Escherichia coli | |
0.204 | - |
5,10-methylene-5,6,7,8-tetrahydrofolyl-(L-Glu)2 | mutant R15E, pH 9.8 | Lacticaseibacillus casei | |
0.23 | - |
ATP | mutant D154A, pH 9.8, cosubstrate 5,6,7,8-tetrahydrofolic acid | Escherichia coli | |
0.23 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant F75A, pH 9.8 | Lacticaseibacillus casei | |
0.23 | - |
ATP | mutant Y414A, pH 9.8 | Lacticaseibacillus casei | |
0.292 | - |
L-Glu | chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, pH 9.8 | Lacticaseibacillus casei | |
0.292 | - |
L-Glu | chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, pH 9.8 | Escherichia coli | |
0.3 | - |
5,6,7,8-tetrahydrofolyl-gamma-(L-Glu)2 | mutant D154A, pH 9.8 | Escherichia coli | |
0.3 | - |
L-Glu | wild-type, pH 9.8 | Escherichia coli | |
0.34 | - |
5,6,7,8-tetrahydrofolic acid | mutant D154A, pH 9.8 | Escherichia coli | |
0.39 | - |
5,6,7,8-tetrahydrofolic acid | mutant T122W, pH 9.8 | Escherichia coli | |
0.4 | - |
5,6,7,8-tetrahydrofolic acid | mutant T122H, pH 9.8 | Escherichia coli | |
0.448 | - |
5,6,7,8-tetrahydrofolic acid | mutant A155H, pH 9.8 | Escherichia coli | |
0.47 | - |
L-Glu | wild-type, pH 9.8 | Lacticaseibacillus casei | |
0.485 | - |
7,8-dihydropteroate | mutant A155H, pH 9.8 | Escherichia coli | |
0.555 | - |
ATP | mutant A155W, pH 9.8 cosubstrate 7,8-dihydropteroate | Escherichia coli | |
0.59 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, pH 9.8 | Lacticaseibacillus casei | |
0.59 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, pH 9.8 | Escherichia coli | |
0.98 | - |
7,8-dihydropteroate | mutant T122H, pH 9.8 | Escherichia coli | |
1.5 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant F121A, pH 9.8 | Lacticaseibacillus casei | |
2.7 | - |
L-Glu | mutant Y414A, pH 9.8 | Lacticaseibacillus casei | |
3.3 | - |
L-Glu | mutant D154A, pH 9.8, cosubstrate 5,6,7,8-tetrahydrofolic acid | Escherichia coli | |
3.4 | - |
ATP | mutant D151A, pH 9.8 | Lacticaseibacillus casei | |
3.4 | - |
ATP | wild-type, pH 9.8 | Lacticaseibacillus casei | |
3.8 | - |
5,10-methylene-5,6,7,8-tetrahydrofolic acid | mutant T119W, pH 9.8 | Lacticaseibacillus casei | |
5.7 | - |
ATP | chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, pH 9.8 | Lacticaseibacillus casei | |
5.7 | - |
ATP | chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, pH 9.8 | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P08192 | - |
- |
Lacticaseibacillus casei | P15925 | - |
- |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.138 | - |
chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Lacticaseibacillus casei |
0.138 | - |
chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Escherichia coli |
0.376 | - |
chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, substrate 7,8-dihydropteroate, pH 9.8 | Lacticaseibacillus casei |
0.376 | - |
chimera containing N-terminal residues 1-81 of Escherichia coli enzyme, residues 72-82 of Lactobacillus casei enzyme, and residues 93-422 of Escherichia coli enzyme, substrate 7,8-dihydropteroate, pH 9.8 | Escherichia coli |
0.912 | - |
chimera containing N-terminal residues 1-71 of Lactobacillus casei enzyme, residues 82-93 of Escherichia coli enzyme, and residues 83-428 of Lactobacillus casei enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Lacticaseibacillus casei |
0.912 | - |
chimera containing N-terminal residues 1-71 of Lactobacillus casei enzyme, residues 82-93 of Escherichia coli enzyme, and residues 83-428 of Lactobacillus casei enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Escherichia coli |
1.3 | - |
chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Lacticaseibacillus casei |
1.3 | - |
chimera containing N-terminal residues 1-298 of Lactobacillus casei enzyme, residues 288-422 of Escherichia coli enzyme, substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Escherichia coli |
4.1 | - |
substrate 7,8-dihydropteroate, pH 9.8 | Escherichia coli |
11.8 | - |
substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Lacticaseibacillus casei |
13.2 | - |
substrate 5,10-methylene-5,6,7,8-tetrahydrofolic acid, pH 9.8 | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + 5,10-methylene-5,6,7,8-tetrahydrofolic acid + L-Glu | - |
Escherichia coli | ADP + phosphate + 5,10-methylene-5,6,7,8-tetrahydrofolyl-L-Glu | - |
? | |
ATP + 5,10-methylene-5,6,7,8-tetrahydrofolic acid + L-glutamate | - |
Lacticaseibacillus casei | ADP + phosphate + 5,10-methylene-5,6,7,8-tetrahydrofolyl-Glu | - |
? | |
ATP + 5,10-methylene-5,6,7,8-tetrahydrofolyl-(L-Glu)2 + L-glutamate | - |
Lacticaseibacillus casei | ADP + phosphate + 5,10-methylene-5,6,7,8-tetrahydrofolyl-(L-Glu)3 | - |
? | |
ATP + 5,6,7,8-tetrahydrofolic acid + L-Glu | - |
Escherichia coli | ADP + phosphate + 5,6,7,8-tetrahydrofolyl-Glu | - |
? | |
ATP + 5,6,7,8-tetrahydrofolyl-gamma-(L-Glu)2 + L-Glu | - |
Escherichia coli | ADP + phosphate + 5,6,7,8-tetrahydrofolyl-gamma-(L-Glu)3 | - |
? | |
ATP + 7,8-dihydropteroate + L-Glu | - |
Escherichia coli | ADP + phosphate + 7,8-dihydropteroyl-L-Glu | - |
? |