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 language | English (US) |
|---|---|
| Pages (from-to) | 16971-16977 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 281 |
| Issue number | 25 |
| DOIs | |
| State | Published - Jun 23 2006 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
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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 journal › Article
}
TY - JOUR
T1 - Purification and initial biochemical characterization of ATP:Cob(I)alamin adenosyltransferase (EutT) enzyme of Salmonella enterica
AU - Buan, Nicole R.
AU - Escalante-Semerena, Jorge C.
PY - 2006/6/23
Y1 - 2006/6/23
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33745203067&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33745203067&partnerID=8YFLogxK
U2 - 10.1074/jbc.M603069200
DO - 10.1074/jbc.M603069200
M3 - Article
C2 - 16636051
AN - SCOPUS:33745203067
VL - 281
SP - 16971
EP - 16977
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 25
ER -