Cloned (Comment) | Organism |
---|---|
QuickChangeXL site-directed mutagenesis performed on wild-type LrpduO in pTEV3 before expression of wild-type and variants in Escherichia coli BL21(DE3) with a rTEV protease-cleavable N-terminal hexa-His tag | Limosilactobacillus reuteri |
Crystallization (Comment) | Organism |
---|---|
mutants D35N (without tag) in complex with ATP and cob(II)alamin and R132K (without tag) in complex with ATP, thin plate crystals, space group P6(3), two monomers in the asymmetric unit, unit cell parameters: a, b: 65A, c: 169A, beta: 90°; vapour-diffusion under anoxic conditions, protein solution (15 mg/ml, containing ATP, hydroxycobalamin and a reducing system of NADH, FMN, and flavodoxin reductase), reservoir solution (incl. 14-16% PEG8000, pH6) | Limosilactobacillus reuteri |
Protein Variants | Comment | Organism |
---|---|---|
D35E/R128K | reduced activity to lower extent than mutation R128K alone | Limosilactobacillus reuteri |
D35N | 230fold decrease in kcat/KM (ATP) most likely due to disruption of salt bridge with residue R128 as observed in crystal structure | Limosilactobacillus reuteri |
D35N/R128K | similar kinetics as mutation D35N alone | Limosilactobacillus reuteri |
D35R/R128D | reciprocal mutation to D35/R128, very high KM values did not allow for kinetic analyses at saturating substrate concentrations | Limosilactobacillus reuteri |
R128K | kinetics similar to wild-type, R128 conserved among PduO-type ACAs, R128 and R128K build salt bridge to residue D35 of adjacent unit, mutation R128W most common in methylmalonic aciduria patients | Limosilactobacillus reuteri |
R132K | 60-80fold decrease in kcat with respect to both substrates, 300fold decrease in kcat/KM (ATP), 3000fold decrease in kcat/KM (cob(I)alamin), identical position of ATP in the crystal structure compared to wild-type, no cob(I)alamin detectable in crystal structure | Limosilactobacillus reuteri |
S129A | mildly affected kcat and KM for both substrated | Limosilactobacillus reuteri |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
cob(I)alamin | substrate inhibition at more than 0.008 mM in presence of subsaturating concentrations of ATP (0.003 mM) | Limosilactobacillus reuteri | |
cobinamide | substrate inhibition at more than 0.005 mM in presence of subsaturating concentrations of ATP (0.003 mM) | Limosilactobacillus reuteri |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | wild-type: KM for CTP and UTP increased relative to ATP | Limosilactobacillus reuteri | |
additional information | - |
additional information | wild-type: KM for GTP and ITP 530-13000fold increased relative to ATP | Limosilactobacillus reuteri | |
0.0016 | - |
2'-deoxy-ATP | - |
Limosilactobacillus reuteri | |
0.0022 | - |
ATP | - |
Limosilactobacillus reuteri | |
1.16 | - |
GTP | - |
Limosilactobacillus reuteri | |
22 | - |
CTP | - |
Limosilactobacillus reuteri | |
29 | - |
ITP | - |
Limosilactobacillus reuteri | |
40 | - |
UTP | - |
Limosilactobacillus reuteri |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Limosilactobacillus reuteri | - |
- |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
2 ATP + 2 cob(II)alamin + a reduced flavoprotein = 2 triphosphate + 2 adenosylcob(III)alamin + an oxidized flavoprotein | nucleophilic attack from reduced Co1+ ion of cob(I)alamin to the C-5 carbon of ATP, ordered substrate binding mechanism with ATP being first and essential for catalysis | Limosilactobacillus reuteri |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2'-deoxy-ATP + cob(I)alamin + H2O | - |
Limosilactobacillus reuteri | ? | - |
? | |
ATP + cob(I)alamin + H2O | 37°C | Limosilactobacillus reuteri | adenosylcobalamin + diphosphate + phosphate | monitoring adenosylcobalamin formation at 388 nm in continous spectrophotometric assay | ? | |
CTP + cob(I)alamin + H2O | 37°C | Limosilactobacillus reuteri | cytidylcobalamin + diphosphate + phosphate | - |
? | |
GTP + cob(I)alamin + H2O | 37°C | Limosilactobacillus reuteri | guanosylcobalamin + diphosphate + phosphate | - |
? | |
ITP + cob(I)alamin + H2O | 37°C | Limosilactobacillus reuteri | inosylcobalamin + diphosphate + phosphate | - |
? | |
additional information | TTP does not serve as substrate | Limosilactobacillus reuteri | ? | - |
? | |
UTP + cob(I)alamin + H2O | 37°C | Limosilactobacillus reuteri | uridylcobalamin + diphosphate + phosphate | - |
? |
Subunits | Comment | Organism |
---|---|---|
trimer | wild-type and mutants D35N and R128K, revealed by gel filtration chromatography | Limosilactobacillus reuteri |
Synonyms | Comment | Organism |
---|---|---|
human-type ACA | - |
Limosilactobacillus reuteri |
human-type ATP: co(I)rrinoid adenosyltransferase | PduO | Limosilactobacillus reuteri |
LrPduO | PduO from Lactobacillus reuteri | Limosilactobacillus reuteri |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
UTP | kcat/KM: 0.048 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
CTP | kcat/KM: 0.38 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ITP | kcat/KM: 0.65 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | kcat/KM: 12000 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
GTP | kcat/KM: 18 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | kcat/KM: 180000 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
2'-deoxy-ATP | kcat/KM: 3300 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant D35E/R128K, kcat/KM: 23 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant D35E/R128K, kcat/KM: 900 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant D35N, kcat/KM: 2800 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant D35N, kcat/KM: 52 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant D35N/R128K, kcat/KM: 32 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant D35N/R128K, kcat/KM: 3800 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant D35R/R128D, kcat/KM: 0.014 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant D35R/R128D, kcat/KM: 5.2 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant R128K, kcat/KM: 12000 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant R128K, kcat/KM: 1800 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant R132K, kcat/KM: 39 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant R132K, kcat/KM: 55 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
ATP | mutant S129A, kcat/KM: 4500 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
cob(I)alamin | mutant S129A, kcat/KM: 65000 1/M*s | Limosilactobacillus reuteri | |
additional information | - |
additional information | wild-type, decreased kcat for CTP and UTP relative to ATP | Limosilactobacillus reuteri | |
additional information | - |
additional information | wild-type, kcat for GTP and ITP is not decreased relative to ATP | Limosilactobacillus reuteri | |
0.00041 | - |
cob(I)alamin | mutant R132K | Limosilactobacillus reuteri | |
0.00065 | - |
cob(I)alamin | mutant D35R/R128D, at subsaturating concentrations of ATP 30 mM ATP and 0.02 mM cob(I)alamin | Limosilactobacillus reuteri | |
0.00195 | - |
UTP | - |
Limosilactobacillus reuteri | |
0.0024 | - |
cob(I)alamin | mutant D35E/R128K | Limosilactobacillus reuteri | |
0.0038 | - |
ATP | mutant S129A | Limosilactobacillus reuteri | |
0.0051 | - |
2'-deoxy-ATP | - |
Limosilactobacillus reuteri | |
0.0062 | - |
ATP | mutant R128K | Limosilactobacillus reuteri | |
0.0068 | - |
cob(I)alamin | mutant D35N | Limosilactobacillus reuteri | |
0.0084 | - |
ATP | mutant R132K | Limosilactobacillus reuteri | |
0.0086 | - |
CTP | - |
Limosilactobacillus reuteri | |
0.011 | - |
cob(I)alamin | mutant R128K | Limosilactobacillus reuteri | |
0.014 | - |
cob(I)alamin | mutant D35N/R128K | Limosilactobacillus reuteri | |
0.015 | - |
cob(I)alamin | mutant S129A | Limosilactobacillus reuteri | |
0.019 | - |
ITP | - |
Limosilactobacillus reuteri | |
0.021 | - |
GTP | - |
Limosilactobacillus reuteri | |
0.024 | - |
cob(I)alamin | - |
Limosilactobacillus reuteri | |
0.026 | - |
ATP | - |
Limosilactobacillus reuteri | |
0.096 | - |
ATP | mutant D35E/R128K | Limosilactobacillus reuteri | |
0.118 | - |
ATP | mutant D35N | Limosilactobacillus reuteri | |
0.41 | - |
ATP | mutant D35N/R128K | Limosilactobacillus reuteri | |
9 | - |
ATP | mutant D35R/R128D, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin | Limosilactobacillus reuteri |