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Literature summary 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)

EC Number Cloned (Comment) 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 Limosilactobacillus reuteri

Crystallization (Commentary)

EC Number Crystallization (Comment) 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) Limosilactobacillus reuteri

Protein Variants

EC Number Protein Variants Comment Organism
2.5.1.17 D35E/R128K reduced activity to lower extent than mutation R128K alone Limosilactobacillus 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 Limosilactobacillus reuteri
2.5.1.17 D35N/R128K similar kinetics as mutation D35N alone Limosilactobacillus 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 Limosilactobacillus 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 Limosilactobacillus 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 Limosilactobacillus reuteri
2.5.1.17 S129A mildly affected kcat and KM for both substrated Limosilactobacillus reuteri

Inhibitors

EC Number Inhibitors Comment 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) Limosilactobacillus reuteri
2.5.1.17 cobinamide substrate inhibition at more than 0.005 mM in presence of subsaturating concentrations of ATP (0.003 mM) Limosilactobacillus reuteri

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.5.1.17 additional information
-
 additional information wild-type: KM for CTP and UTP increased relative to ATP Limosilactobacillus reuteri
2.5.1.17 additional information
-
 additional information wild-type: KM for GTP and ITP 530-13000fold increased relative to ATP Limosilactobacillus reuteri
2.5.1.17 0.0016
-
 2'-deoxy-ATP
-
 Limosilactobacillus reuteri
2.5.1.17 0.0022
-
 ATP
-
 Limosilactobacillus reuteri
2.5.1.17 1.16
-
 GTP
-
 Limosilactobacillus reuteri
2.5.1.17 22
-
 CTP
-
 Limosilactobacillus reuteri
2.5.1.17 29
-
 ITP
-
 Limosilactobacillus reuteri
2.5.1.17 40
-
 UTP
-
 Limosilactobacillus reuteri

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.17 Limosilactobacillus reuteri
-
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
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 Limosilactobacillus reuteri
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.5.1.17 2'-deoxy-ATP + cob(I)alamin + H2O
-
 Limosilactobacillus reuteri ?
-
 ?
2.5.1.17 ATP + cob(I)alamin + H2O 37°C Limosilactobacillus 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 Limosilactobacillus reuteri cytidylcobalamin + diphosphate + phosphate
-
 ?
2.5.1.17 GTP + cob(I)alamin + H2O 37°C Limosilactobacillus reuteri guanosylcobalamin + diphosphate + phosphate
-
 ?
2.5.1.17 ITP + cob(I)alamin + H2O 37°C Limosilactobacillus reuteri inosylcobalamin + diphosphate + phosphate
-
 ?
2.5.1.17 additional information TTP does not serve as substrate Limosilactobacillus reuteri ?
-
 ?
2.5.1.17 UTP + cob(I)alamin + H2O 37°C Limosilactobacillus reuteri uridylcobalamin + diphosphate + phosphate
-
 ?

Subunits

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

Synonyms

EC Number Synonyms Comment Organism
2.5.1.17 human-type ACA
-
 Limosilactobacillus reuteri
2.5.1.17 human-type ATP: co(I)rrinoid adenosyltransferase PduO Limosilactobacillus reuteri
2.5.1.17 LrPduO PduO from Lactobacillus reuteri Limosilactobacillus reuteri

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.5.1.17 additional information
-
 UTP kcat/KM: 0.048 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 CTP kcat/KM: 0.38 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ITP kcat/KM: 0.65 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP kcat/KM: 12000 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 GTP kcat/KM: 18 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 cob(I)alamin kcat/KM: 180000 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 2'-deoxy-ATP kcat/KM: 3300 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP mutant D35E/R128K, kcat/KM: 23 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 cob(I)alamin mutant D35E/R128K, kcat/KM: 900 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 cob(I)alamin mutant D35N, kcat/KM: 2800 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP mutant D35N, kcat/KM: 52 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP mutant D35N/R128K, kcat/KM: 32 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 cob(I)alamin mutant D35N/R128K, kcat/KM: 3800 1/M*s Limosilactobacillus reuteri
2.5.1.17 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
2.5.1.17 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
2.5.1.17 additional information
-
 cob(I)alamin mutant R128K, kcat/KM: 12000 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP mutant R128K, kcat/KM: 1800 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP mutant R132K, kcat/KM: 39 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 cob(I)alamin mutant R132K, kcat/KM: 55 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 ATP mutant S129A, kcat/KM: 4500 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 cob(I)alamin mutant S129A, kcat/KM: 65000 1/M*s Limosilactobacillus reuteri
2.5.1.17 additional information
-
 additional information wild-type, decreased kcat for CTP and UTP relative to ATP Limosilactobacillus reuteri
2.5.1.17 additional information
-
 additional information wild-type, kcat for GTP and ITP is not decreased relative to ATP Limosilactobacillus reuteri
2.5.1.17 0.00041
-
 cob(I)alamin mutant R132K Limosilactobacillus 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 Limosilactobacillus reuteri
2.5.1.17 0.00195
-
 UTP
-
 Limosilactobacillus reuteri
2.5.1.17 0.0024
-
 cob(I)alamin mutant D35E/R128K Limosilactobacillus reuteri
2.5.1.17 0.0038
-
 ATP mutant S129A Limosilactobacillus reuteri
2.5.1.17 0.0051
-
 2'-deoxy-ATP
-
 Limosilactobacillus reuteri
2.5.1.17 0.0062
-
 ATP mutant R128K Limosilactobacillus reuteri
2.5.1.17 0.0068
-
 cob(I)alamin mutant D35N Limosilactobacillus reuteri
2.5.1.17 0.0084
-
 ATP mutant R132K Limosilactobacillus reuteri
2.5.1.17 0.0086
-
 CTP
-
 Limosilactobacillus reuteri
2.5.1.17 0.011
-
 cob(I)alamin mutant R128K Limosilactobacillus reuteri
2.5.1.17 0.014
-
 cob(I)alamin mutant D35N/R128K Limosilactobacillus reuteri
2.5.1.17 0.015
-
 cob(I)alamin mutant S129A Limosilactobacillus reuteri
2.5.1.17 0.019
-
 ITP
-
 Limosilactobacillus reuteri
2.5.1.17 0.021
-
 GTP
-
 Limosilactobacillus reuteri
2.5.1.17 0.024
-
 cob(I)alamin
-
 Limosilactobacillus reuteri
2.5.1.17 0.026
-
 ATP
-
 Limosilactobacillus reuteri
2.5.1.17 0.096
-
 ATP mutant D35E/R128K Limosilactobacillus reuteri
2.5.1.17 0.118
-
 ATP mutant D35N Limosilactobacillus reuteri
2.5.1.17 0.41
-
 ATP mutant D35N/R128K Limosilactobacillus reuteri
2.5.1.17 9
-
 ATP mutant D35R/R128D, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin Limosilactobacillus reuteri