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Literature summary for 2.5.1.17 extracted from

  • Mera, P.E.; St Maurice, M.; Rayment, I.; Escalante-Semerena, J.C.
    Structural and functional analyses of the human-type corrinoid adenosyltransferase (PduO) from Lactobacillus reuteri (2007), Biochemistry, 46, 13829-13836.
    View publication on PubMed

Cloned(Commentary)

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 (Commentary)

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

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

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 [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

Organism UniProt Comment Textmining
Limosilactobacillus reuteri
-
-
-

Reaction

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 and Products (Substrate)

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

Subunits Comment Organism
trimer wild-type and mutants D35N and R128K, revealed by gel filtration chromatography Limosilactobacillus reuteri

Synonyms

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 [1/s]

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