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Literature summary extracted from

  • Otte, M.M.; Woodson, J.D.; Escalante-Semerena, J.C.
    The thiamine kinase (YcfN) enzyme plays a minor but significant role in cobinamide salvaging in Salmonella enterica (2007), J. Bacteriol., 189, 7310-7315.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.89 expressed in Escherichia coli Salmonella enterica
2.7.1.156 A chromosomal in-frame deletion of the cobU gene is constructed in strain TR6583. Salmonella enterica subsp. enterica serovar Typhimurium
2.7.7.62 in pET-16b (pCLK740) for expression with N-terminal deca-His-tag in Salmonella enterica serovar Typhimurium LT2, strain JE8407 lacking endogenous CobU and YcfN Escherichia coli

General Stability

EC Number General Stability Organism
2.7.7.62 no soluble N-terminal His-tagged protein detectable after immobilized metal affinity chromatography from over-expressing Salmonella enterica serovar Typhimurium LT2 Escherichia coli

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.1.89 34000
-
 SDS-PAGE, His-tagged enzyme Salmonella enterica
2.7.7.62 34000
-
 SDS-PAGE Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.156 adenosylcobinamide + ATP Salmonella enterica subsp. enterica serovar Typhimurium
-
 adenosylcobinamide phosphate + ADP
-
 ir
2.7.1.156 adenosylcobinamide + GTP Salmonella enterica subsp. enterica serovar Typhimurium
-
 adenosylcobinamide phosphate + GDP
-
 ir
2.7.1.156 adenosylcobinamide phosphate + GTP Salmonella enterica subsp. enterica serovar Typhimurium
-
 adenosylcobinamide-GDP + diphosphate
-
 ir
2.7.1.156 additional information Salmonella enterica subsp. enterica serovar Typhimurium CobU enzyme of the nucleotide loop assembly pathway is a bifunctional enzyme with adenosylcobinamide and adenosylcobinamide-phosphate guanylyltransferase activity, important for de novo synthesis and for cobinamide salvaging ?
-
 ?
2.7.6.2 additional information Salmonella enterica the YcfN protein acts as a nonspecific adenosylcobinamide kinase enzyme, which is not associated with adenosylcobalamine biosynthesis, but it allows a CobU-deficient strain of serovar Typhimurium to synthesize enough adenosylcobalamine from cobinamide so that the cell can grow. YcfN is required for the phosphorylation of thiamine in the thiamine salvaging pathway of Escherichia coli ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.89 Salmonella enterica
-
-
-
2.7.1.156 Salmonella enterica subsp. enterica serovar Typhimurium
-
 Typhimurium LT2
-
2.7.6.2 Salmonella enterica
-
 serovar typhimurium LT2
-
2.7.7.62 Escherichia coli
-
-
-
2.7.7.62 Salmonella enterica
-
 serovar typhimurium LT2
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.156 CobU protein is overproduced and purified. Cells from a 4-liter culture of the overproducing strain are lysed using a French press. After the addition of ammonium sulfate, fast protein liquid chromatography is used to isolate CobU protein from the clarified cell extract in two additional steps, hydrophobic interaction chromatography and dye ligand chromatography. Protein concentration was determined by the Bradford method. Salmonella enterica subsp. enterica serovar Typhimurium
2.7.7.62 tandem hydrophobic interaction chromatography on two HiTrap Phenyl FastFlow columns, dialysation, and dye ligand chromatography on HiTrap Blue HP column (elution with 2 M NaCl) Salmonella enterica

Source Tissue

EC Number Source Tissue Comment Organism Textmining
2.7.1.156 cell culture
-
 Salmonella enterica subsp. enterica serovar Typhimurium
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.89 ATP + adenosylcobinamide
-
 Salmonella enterica ADP + adenosylcobinamide monophosphate
-
 ?
2.7.1.89 ATP + thiamine
-
 Salmonella enterica ADP + thiamine monophosphate
-
 ?
2.7.1.156 adenosylcobinamide + ATP
-
 Salmonella enterica subsp. enterica serovar Typhimurium adenosylcobinamide phosphate + ADP
-
 ir
2.7.1.156 adenosylcobinamide + GTP
-
 Salmonella enterica subsp. enterica serovar Typhimurium adenosylcobinamide phosphate + GDP
-
 ir
2.7.1.156 adenosylcobinamide phosphate + GTP
-
 Salmonella enterica subsp. enterica serovar Typhimurium adenosylcobinamide-GDP + diphosphate
-
 ir
2.7.1.156 additional information CobU enzyme of the nucleotide loop assembly pathway is a bifunctional enzyme with adenosylcobinamide and adenosylcobinamide-phosphate guanylyltransferase activity, important for de novo synthesis and for cobinamide salvaging Salmonella enterica subsp. enterica serovar Typhimurium ?
-
 ?
2.7.6.2 additional information the YcfN protein acts as a nonspecific adenosylcobinamide kinase enzyme, which is not associated with adenosylcobalamine biosynthesis, but it allows a CobU-deficient strain of serovar Typhimurium to synthesize enough adenosylcobalamine from cobinamide so that the cell can grow. YcfN is required for the phosphorylation of thiamine in the thiamine salvaging pathway of Escherichia coli Salmonella enterica ?
-
 ?
2.7.7.62 adenosylcobinamide-GDP + diphosphate 2 mM ATP, 5 mM MgCl2, 16 h, pH 8, 37°C Salmonella enterica GTP + adenosylcobinamide phosphate reaction stop by 25 mM KCN and 10 min heating at 80°C, analysis by mass spectrometry ?
2.7.7.62 adenosylcobinamide-GDP + diphosphate over-expressing cell extracts, 2 mM ATP, 5 mM MgCl2, 16 h, pH 8, 37°C Escherichia coli GTP + adenosylcobinamide phosphate reaction stop by 25 mM KCN and 10 min heating at 80°C, analysis by mass spectrometry ?

Synonyms

EC Number Synonyms Comment Organism
2.7.1.89 YcfN the enzyme has also ATP:adenosylcobinamide kinase activity Salmonella enterica
2.7.1.156 ATP/GTP:AdoCbi kinase
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 CobU
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 GTP:adenosylcobinamide-phosphate guanylyltransferase
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 GTP:AdoCbi-P kinase
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 GTP:AdoCbi-phosphate guanylyltransferase
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 NTP:adenosylcobinamide kinase
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 NTP:AdoCbi kinase
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.6.2 YcfN
-
 Salmonella enterica
2.7.7.62 CobU
-
 Salmonella enterica
2.7.7.62 GTP: adenosylcobinamide-phosphate guanylyltransferase GTP: AdoCbi-P guanylyltransferase Salmonella enterica
2.7.7.62 NTP: adenosylcobinamide kinase NTP: AdoCbi kinase Salmonella enterica
2.7.7.62 thiamine kinase
-
 Escherichia coli
2.7.7.62 YcfN
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.1.156 37
-
 assay at Salmonella enterica subsp. enterica serovar Typhimurium

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.1.156 8
-
 assay at Salmonella enterica subsp. enterica serovar Typhimurium

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.89 ATP
-
 Salmonella enterica
2.7.1.156 ATP
-
 Salmonella enterica subsp. enterica serovar Typhimurium
2.7.1.156 GTP
-
 Salmonella enterica subsp. enterica serovar Typhimurium