BRENDA - Enzyme Database

Structural and functional analyses of the human-type corrinoid adenosyltransferase (PduO) from Lactobacillus reuteri

Mera, P.E.; St Maurice, M.; Rayment, I.; Escalante-Semerena, J.C.; Biochemistry 46, 13829-13836 (2007)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
2.5.1.17
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
Lactobacillus reuteri
Crystallization (Commentary)
EC Number
Crystallization
Organism
2.5.1.17
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)
Lactobacillus reuteri
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.5.1.17
D35E/R128K
reduced activity to lower extent than mutation R128K alone
Lactobacillus reuteri
2.5.1.17
D35N
230fold decrease in kcat/KM (ATP) most likely due to disruption of salt bridge with residue R128 as observed in crystal structure
Lactobacillus reuteri
2.5.1.17
D35N/R128K
similar kinetics as mutation D35N alone
Lactobacillus reuteri
2.5.1.17
D35R/R128D
reciprocal mutation to D35/R128, very high KM values did not allow for kinetic analyses at saturating substrate concentrations
Lactobacillus reuteri
2.5.1.17
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
Lactobacillus reuteri
2.5.1.17
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
Lactobacillus reuteri
2.5.1.17
S129A
mildly affected kcat and KM for both substrated
Lactobacillus reuteri
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.5.1.17
cob(I)alamin
substrate inhibition at more than 0.008 mM in presence of subsaturating concentrations of ATP (0.003 mM)
Lactobacillus reuteri
2.5.1.17
cobinamide
substrate inhibition at more than 0.005 mM in presence of subsaturating concentrations of ATP (0.003 mM)
Lactobacillus reuteri
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.5.1.17
additional information
-
additional information
wild-type: KM for CTP and UTP increased relative to ATP; wild-type: KM for GTP and ITP 530-13000fold increased relative to ATP
Lactobacillus reuteri
2.5.1.17
0.0016
-
2'-deoxy-ATP
-
Lactobacillus reuteri
2.5.1.17
0.0022
-
ATP
-
Lactobacillus reuteri
2.5.1.17
1.16
-
GTP
-
Lactobacillus reuteri
2.5.1.17
22
-
CTP
-
Lactobacillus reuteri
2.5.1.17
29
-
ITP
-
Lactobacillus reuteri
2.5.1.17
40
-
UTP
-
Lactobacillus reuteri
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.5.1.17
Lactobacillus reuteri
-
-
-
Reaction
EC Number
Reaction
Commentary
Organism
2.5.1.17
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
Lactobacillus reuteri
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.5.1.17
2'-deoxy-ATP + cob(I)alamin + H2O
-
690887
Lactobacillus reuteri
?
-
-
-
?
2.5.1.17
ATP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
adenosylcobalamin + diphosphate + phosphate
monitoring adenosylcobalamin formation at 388 nm in continous spectrophotometric assay
-
-
?
2.5.1.17
CTP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
cytidylcobalamin + diphosphate + phosphate
-
-
-
?
2.5.1.17
GTP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
guanosylcobalamin + diphosphate + phosphate
-
-
-
?
2.5.1.17
ITP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
inosylcobalamin + diphosphate + phosphate
-
-
-
?
2.5.1.17
additional information
TTP does not serve as substrate
690887
Lactobacillus reuteri
?
-
-
-
-
2.5.1.17
UTP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
uridylcobalamin + diphosphate + phosphate
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
2.5.1.17
trimer
wild-type and mutants D35N and R128K, revealed by gel filtration chromatography
Lactobacillus reuteri
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.5.1.17
additional information
-
2'-deoxy-ATP
kcat/KM: 3300 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
ATP
kcat/KM: 12000 1/M*s; mutant D35E/R128K, kcat/KM: 23 1/M*s; mutant D35N, kcat/KM: 52 1/M*s; mutant D35N/R128K, kcat/KM: 32 1/M*s; mutant D35R/R128D, kcat/KM: 0.014 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin; mutant R128K, kcat/KM: 1800 1/M*s; mutant R132K, kcat/KM: 39 1/M*s; mutant S129A, kcat/KM: 4500 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
cob(I)alamin
kcat/KM: 180000 1/M*s; mutant D35E/R128K, kcat/KM: 900 1/M*s; mutant D35N, kcat/KM: 2800 1/M*s; mutant D35N/R128K, kcat/KM: 3800 1/M*s; mutant D35R/R128D, kcat/KM: 5.2 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin; mutant R128K, kcat/KM: 12000 1/M*s; mutant R132K, kcat/KM: 55 1/M*s; mutant S129A, kcat/KM: 65000 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
CTP
kcat/KM: 0.38 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
GTP
kcat/KM: 18 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
ITP
kcat/KM: 0.65 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
additional information
wild-type, decreased kcat for CTP and UTP relative to ATP; wild-type, kcat for GTP and ITP is not decreased relative to ATP
Lactobacillus reuteri
2.5.1.17
additional information
-
UTP
kcat/KM: 0.048 1/M*s
Lactobacillus reuteri
2.5.1.17
0.00041
-
cob(I)alamin
mutant R132K
Lactobacillus reuteri
2.5.1.17
0.00065
-
cob(I)alamin
mutant D35R/R128D, at subsaturating concentrations of ATP 30 mM ATP and 0.02 mM cob(I)alamin
Lactobacillus reuteri
2.5.1.17
0.00195
-
UTP
-
Lactobacillus reuteri
2.5.1.17
0.0024
-
cob(I)alamin
mutant D35E/R128K
Lactobacillus reuteri
2.5.1.17
0.0038
-
ATP
mutant S129A
Lactobacillus reuteri
2.5.1.17
0.0051
-
2'-deoxy-ATP
-
Lactobacillus reuteri
2.5.1.17
0.0062
-
ATP
mutant R128K
Lactobacillus reuteri
2.5.1.17
0.0068
-
cob(I)alamin
mutant D35N
Lactobacillus reuteri
2.5.1.17
0.0084
-
ATP
mutant R132K
Lactobacillus reuteri
2.5.1.17
0.0086
-
CTP
-
Lactobacillus reuteri
2.5.1.17
0.011
-
cob(I)alamin
mutant R128K
Lactobacillus reuteri
2.5.1.17
0.014
-
cob(I)alamin
mutant D35N/R128K
Lactobacillus reuteri
2.5.1.17
0.015
-
cob(I)alamin
mutant S129A
Lactobacillus reuteri
2.5.1.17
0.019
-
ITP
-
Lactobacillus reuteri
2.5.1.17
0.021
-
GTP
-
Lactobacillus reuteri
2.5.1.17
0.024
-
cob(I)alamin
-
Lactobacillus reuteri
2.5.1.17
0.026
-
ATP
-
Lactobacillus reuteri
2.5.1.17
0.096
-
ATP
mutant D35E/R128K
Lactobacillus reuteri
2.5.1.17
0.118
-
ATP
mutant D35N
Lactobacillus reuteri
2.5.1.17
0.41
-
ATP
mutant D35N/R128K
Lactobacillus reuteri
2.5.1.17
9
-
ATP
mutant D35R/R128D, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin
Lactobacillus reuteri
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.5.1.17
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
Lactobacillus reuteri
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.5.1.17
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)
Lactobacillus reuteri
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.5.1.17
D35E/R128K
reduced activity to lower extent than mutation R128K alone
Lactobacillus reuteri
2.5.1.17
D35N
230fold decrease in kcat/KM (ATP) most likely due to disruption of salt bridge with residue R128 as observed in crystal structure
Lactobacillus reuteri
2.5.1.17
D35N/R128K
similar kinetics as mutation D35N alone
Lactobacillus reuteri
2.5.1.17
D35R/R128D
reciprocal mutation to D35/R128, very high KM values did not allow for kinetic analyses at saturating substrate concentrations
Lactobacillus reuteri
2.5.1.17
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
Lactobacillus reuteri
2.5.1.17
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
Lactobacillus reuteri
2.5.1.17
S129A
mildly affected kcat and KM for both substrated
Lactobacillus reuteri
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.5.1.17
cob(I)alamin
substrate inhibition at more than 0.008 mM in presence of subsaturating concentrations of ATP (0.003 mM)
Lactobacillus reuteri
2.5.1.17
cobinamide
substrate inhibition at more than 0.005 mM in presence of subsaturating concentrations of ATP (0.003 mM)
Lactobacillus reuteri
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.5.1.17
additional information
-
additional information
wild-type: KM for CTP and UTP increased relative to ATP; wild-type: KM for GTP and ITP 530-13000fold increased relative to ATP
Lactobacillus reuteri
2.5.1.17
0.0016
-
2'-deoxy-ATP
-
Lactobacillus reuteri
2.5.1.17
0.0022
-
ATP
-
Lactobacillus reuteri
2.5.1.17
1.16
-
GTP
-
Lactobacillus reuteri
2.5.1.17
22
-
CTP
-
Lactobacillus reuteri
2.5.1.17
29
-
ITP
-
Lactobacillus reuteri
2.5.1.17
40
-
UTP
-
Lactobacillus reuteri
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.5.1.17
2'-deoxy-ATP + cob(I)alamin + H2O
-
690887
Lactobacillus reuteri
?
-
-
-
?
2.5.1.17
ATP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
adenosylcobalamin + diphosphate + phosphate
monitoring adenosylcobalamin formation at 388 nm in continous spectrophotometric assay
-
-
?
2.5.1.17
CTP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
cytidylcobalamin + diphosphate + phosphate
-
-
-
?
2.5.1.17
GTP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
guanosylcobalamin + diphosphate + phosphate
-
-
-
?
2.5.1.17
ITP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
inosylcobalamin + diphosphate + phosphate
-
-
-
?
2.5.1.17
additional information
TTP does not serve as substrate
690887
Lactobacillus reuteri
?
-
-
-
-
2.5.1.17
UTP + cob(I)alamin + H2O
37°C
690887
Lactobacillus reuteri
uridylcobalamin + diphosphate + phosphate
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
2.5.1.17
trimer
wild-type and mutants D35N and R128K, revealed by gel filtration chromatography
Lactobacillus reuteri
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.5.1.17
additional information
-
2'-deoxy-ATP
kcat/KM: 3300 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
ATP
kcat/KM: 12000 1/M*s; mutant D35E/R128K, kcat/KM: 23 1/M*s; mutant D35N, kcat/KM: 52 1/M*s; mutant D35N/R128K, kcat/KM: 32 1/M*s; mutant D35R/R128D, kcat/KM: 0.014 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin; mutant R128K, kcat/KM: 1800 1/M*s; mutant R132K, kcat/KM: 39 1/M*s; mutant S129A, kcat/KM: 4500 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
cob(I)alamin
kcat/KM: 180000 1/M*s; mutant D35E/R128K, kcat/KM: 900 1/M*s; mutant D35N, kcat/KM: 2800 1/M*s; mutant D35N/R128K, kcat/KM: 3800 1/M*s; mutant D35R/R128D, kcat/KM: 5.2 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin; mutant R128K, kcat/KM: 12000 1/M*s; mutant R132K, kcat/KM: 55 1/M*s; mutant S129A, kcat/KM: 65000 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
CTP
kcat/KM: 0.38 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
GTP
kcat/KM: 18 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
ITP
kcat/KM: 0.65 1/M*s
Lactobacillus reuteri
2.5.1.17
additional information
-
additional information
wild-type, decreased kcat for CTP and UTP relative to ATP; wild-type, kcat for GTP and ITP is not decreased relative to ATP
Lactobacillus reuteri
2.5.1.17
additional information
-
UTP
kcat/KM: 0.048 1/M*s
Lactobacillus reuteri
2.5.1.17
0.00041
-
cob(I)alamin
mutant R132K
Lactobacillus reuteri
2.5.1.17
0.00065
-
cob(I)alamin
mutant D35R/R128D, at subsaturating concentrations of ATP 30 mM ATP and 0.02 mM cob(I)alamin
Lactobacillus reuteri
2.5.1.17
0.00195
-
UTP
-
Lactobacillus reuteri
2.5.1.17
0.0024
-
cob(I)alamin
mutant D35E/R128K
Lactobacillus reuteri
2.5.1.17
0.0038
-
ATP
mutant S129A
Lactobacillus reuteri
2.5.1.17
0.0051
-
2'-deoxy-ATP
-
Lactobacillus reuteri
2.5.1.17
0.0062
-
ATP
mutant R128K
Lactobacillus reuteri
2.5.1.17
0.0068
-
cob(I)alamin
mutant D35N
Lactobacillus reuteri
2.5.1.17
0.0084
-
ATP
mutant R132K
Lactobacillus reuteri
2.5.1.17
0.0086
-
CTP
-
Lactobacillus reuteri
2.5.1.17
0.011
-
cob(I)alamin
mutant R128K
Lactobacillus reuteri
2.5.1.17
0.014
-
cob(I)alamin
mutant D35N/R128K
Lactobacillus reuteri
2.5.1.17
0.015
-
cob(I)alamin
mutant S129A
Lactobacillus reuteri
2.5.1.17
0.019
-
ITP
-
Lactobacillus reuteri
2.5.1.17
0.021
-
GTP
-
Lactobacillus reuteri
2.5.1.17
0.024
-
cob(I)alamin
-
Lactobacillus reuteri
2.5.1.17
0.026
-
ATP
-
Lactobacillus reuteri
2.5.1.17
0.096
-
ATP
mutant D35E/R128K
Lactobacillus reuteri
2.5.1.17
0.118
-
ATP
mutant D35N
Lactobacillus reuteri
2.5.1.17
0.41
-
ATP
mutant D35N/R128K
Lactobacillus reuteri
2.5.1.17
9
-
ATP
mutant D35R/R128D, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin
Lactobacillus reuteri