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2.5.1.17: corrinoid adenosyltransferase

This is an abbreviated version!
For detailed information about corrinoid adenosyltransferase, go to the full flat file.

Word Map on EC 2.5.1.17

Reaction

2 ATP + 2 cob(II)alamin +

a reduced flavoprotein
= 2 triphosphate + 2 adenosylcob(III)alamin +
an oxidized flavoprotein

Synonyms

ACAT, adenosyltransferase, vitamin B12s, aquacob(I)alamin adenosyltransferase, aquocob(I)alamin adenosyltransferase, ATP: cobalamin adenosyltransferase, ATP:co(I)rrinoid adenosyltransferase, ATP:cob(I)alamin adenosyltransferase, ATP:cob(I)alamin Cobeta-adenosyltransferase, ATP:cob(I)alamin transferase (ATR), ATP:Cob(I)alaminadenosyltransferase, ATP:cobalamin adenosyltransferase, ATP:cobalt(I) corrinoid adenosyltransferase, ATP:corrinoid adenosyltransferase, ATP:corrinoid adenosyltransferase PduO, ATR, cob(I)alamin adenosyltransferase, cob(I)yrinic acid a,c-diamide adenosyltransferase, CobA, CobA-type ATP:Co(I)rrinoid adenosyltransferase, cobalamin adenosyltransferase, CobAMm, cobO mutant SVQ336, cobO mutant SVQ524, cobO mutantSVQ336, EC 1.16.8.1, EutT, EutT adenosyltransferase, hATR, homologeous to PduO-type ATP: cob(I)alamin adenosyltransferase, human adenosyltransferase, human ATP: cob(I)alamin adenosyltransferase, human-type ACA, human-type ATP: co(I)rrinoid adenosyltransferase, human-type ATP: cob(I)alamin adenosyltransferase, LrPduO, methylmalonic aciduria type B, MMAB, MMAB protein, PduO, PduO adenosyltransferase, PduO enzyme, PduO protein, PduO-type ACA, PduO-type adenosine 5'-triphosphate:corrinoid adenosyltransferase, PduO-type ATP: cobalamin adenosyltransferase, PduO-type ATP:Co(I)rrinoid adenosyltransferase, PduO-type ATP:cob(I)alamin adenosyltransferase, PduO-type ATP:corrinoid adenosyltransferase, PduO-type corrinoid adenosyltransferase, ST1454, ST2180, TA1434, vitamin B12s adenosyltransferase

ECTree

     2 Transferases
         2.5 Transferring alkyl or aryl groups, other than methyl groups
             2.5.1 Transferring alkyl or aryl groups, other than methyl groups (only sub-subclass identified to date)
                2.5.1.17 corrinoid adenosyltransferase

Purification

Purification on EC 2.5.1.17 - corrinoid adenosyltransferase

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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
2 recombinant polymorphic variants 239K and 239M of the enzyme from Escherichia coli in a multistep procedure to homogeneity, 4.5- and 5.4fold, respectively
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after expression at 16°C, lysis with French pressure cell, pH 7.5, and centrifugation
ammonium sulfate precipitation followed by two chromatographic steps
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centrifugation and SDS-PAGE
centrifugation, Mono Q chromatography and gel filtration
from bacterial lysate by heat treatment (30 min, 70°C) and chromatography: HiTrap Phenyl FF column (2-0 M NaCl gradient, pH 8) and HiLoad 26/60 Superdex 75-pg column (pH 8), for the selenomethionine derivative: HiTrap QXL column (0-2 M NaCl gradient, pH 9) and HiLoad 26/60 Superdex 75-pg column (pH 8)
from bacterial lysate by heat treatment (30 min, 70°C) and chromatography: HiTrap QXL column (0-2 M NaCl gradient, pH9) and HiLoad 26/60 Superdex 75-pg column (pH8)
from crude lysate by HiTrap Ni2+-chelating affinity chromatography (gradient elution), gel filtration on HiLoad 16/60 Superdex 200 (pH7.9, 4 mM dihiothreitol), concentration to 15 mg/ml by ultrafiltration, yield: 45 mg pure protein per litre culture
HisTrap column chromatography and Sephadex G-25 gel filtration
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HisTrap nickel affinity column chromatography
native enzyme by anion exchange chromatography, dialysis, and hydrophobic interaction chromatography, recombinant enzyme from Escherichia coli strain BL21(DE3)
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nickel-chelating HiTrap chromatography (elution with 1 M imidazole), HiLoad 16/60 Superdex 200 gel filtration chromatography (2 mM dithiothreitol), concentration to 13 mg/ml by ultracentrifugation
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partial purification of the enzyme is achieved from ribosomal fraction by salt extraction and ammonium sulfate fractionation, followed by two chromatographic steps
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protamine sulfate, heat treatment and chromatography on Sephadex G-25 and DEAE-cellulose
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)
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recombinant His-tagged EutT from Escherichia coli to 70% purity
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
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recombinant His-tagged wild-type and mutant PduOs from Escherichia coli by nickel affinity chromatography to over 99% homogeneity
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recombinant maltose-binding-protein or GST fusion wild-type and mutant enzymes from Escherichia coli strain DH5alpha by amylose and glutathione affinity chromatography, respectively
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recombinant PduO from Escherichia coli strain BL21 (DE3) cell culture medium
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recombinant solubilized enzyme from Escherichia coli 1.4fold to homogeneity, partial purification of recombinant truncated mutants, recombinant N-terminal domain to homogeneity
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recombinant tagless, wild-type CobA, and His7-tagged mutant enzymes by nickel affinity chromatography, the His-tag is cleaved off by rTEV protease
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using a HisTrap FF column
using a HiTrap SP HP FPLC column and gel filtration
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using Ni-NTA chromatography
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