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

The Journal of Biological Chemistry
Nicole R Buan, J C Escalante-Semerena

Abstract

ATP:cob(I)alamin adenosyltransferase (EutT) of Salmonella enterica was overproduced and enriched to approximately 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 (approximately 70% homogeneous) was obtained after treatment with detergent. Under conditions in which cobalamin (Cbl) was saturating, Km(ATP) = 10 microm, kcat = 0.03 s(-1), and Vmax = 54.5 nm min(-1). Similarly, under conditions in which MgATP was saturating, Km(Cbl) = 4.1 microm, 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 > or =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 beta-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...Continue Reading

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Citations

Aug 3, 2010·Applied Microbiology and Biotechnology·Paola E Mera, Jorge C Escalante-Semerena
Sep 29, 1993·Biochimica Et Biophysica Acta·L M WorshamM L Ernst-Fonberg
Jul 30, 2011·Metallomics : Integrated Biometal Science·Lee Macomber, Robert P Hausinger
Nov 26, 2009·The Journal of Biological Chemistry·Paola E Mera, Jorge C Escalante-Semerena
Dec 7, 2007·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Pilar Ruiz-SánchezRoger Alberto
Aug 9, 2012·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Manoj KumarPawel M Kozlowski
Feb 18, 2016·Journal of the American Chemical Society·Ivan G PallaresThomas C Brunold
Sep 4, 2014·Microbiology and Molecular Biology Reviews : MMBR·Chiranjit ChowdhuryThomas A Bobik
Jan 25, 2020·International Journal of Environmental Research and Public Health·Giuseppe GenchiAlessia Catalano
Sep 1, 2008·EcoSal Plus·Jorge C Escalante-Semerena, Martin J Warren
Oct 9, 2020·EcoSal Plus·Katie L StewartThomas A Bobik

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