Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica

Nicole R. Buan, Jorge C. Escalante-Semerena

Research output: Contribution to journalArticle

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Abstract

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to ∼70% homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (∼70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 μM, kcat = 0.03 s-1, and Vmax = 54.5 nM min-1. Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 μM, kcat = 0.06 s-1, and Vmax = 105 nM min -1. Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was ≥50-fold higher with ATP versus GTP, and EutT retained 80% of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the β-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PP i and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.

Original languageEnglish (US)
Pages (from-to)16971-16977
Number of pages7
JournalJournal of Biological Chemistry
Volume281
Issue number25
DOIs
StatePublished - Jun 23 2006

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Salmonella
Salmonella enterica
Purification
Adenosine Triphosphate
Enzymes
Vitamin B 12
Nucleotides
Flavin Mononucleotide
Housekeeping
Proteins
Adenine
Substrates
Adenosine Monophosphate
Operon
Guanosine Triphosphate
Cell Extracts
Kinetic parameters
Detergents
NAD
Adenosine

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica. / Buan, Nicole R.; Escalante-Semerena, Jorge C.

In: Journal of Biological Chemistry, Vol. 281, No. 25, 23.06.2006, p. 16971-16977.

Research output: Contribution to journalArticle

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abstract = "ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to ∼70{\%} homogeneity, and its basic kinetic parameters were determined. Abundant amounts of EutT protein were produced, but all of it remained insoluble. Soluble active EutT protein (∼70{\%} homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 μM, kcat = 0.03 s-1, and Vmax = 54.5 nM min-1. Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 μM, kcat = 0.06 s-1, and Vmax = 105 nM min -1. Unlike other ATP:co(I)rrinoid adenosyltransferases in the cell (i.e. CobA and PduO), EutT activity was ≥50-fold higher with ATP versus GTP, and EutT retained 80{\%} of its activity with ADP substituted for ATP and was completely inactive with AMP as substrate, indicating that the enzyme requires the β-phosphate group of the nucleotide substrate. The data suggest that the amino group of adenine might play a role in nucleotide recognition and/or binding. Unlike the housekeeping CobA enzyme, EutT was not inhibited by inorganic tripolyphosphate (PPPi). Results from 31P NMR spectroscopy studies identified PPi and Pi as by-products of the EutT reaction. In the absence of Cbl, EutT cleaved ATP into adenosine and PPPi, suggesting that PPPi is broken down into PP i and Pi. Electron transfer protein partners for EutT were not encoded by the eut operon. EutT-dependent activity was detected in cell-free extracts of cobA strains enriched for EutT when FMN and NADH were used to reduce cob(III)alamin to cob(I)alamin.",
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