The eutT gene of Salmonella enterica Encodes an oxygen-labile, metal-containing ATP:corrinoid adenosyltransferase enzyme

Abstract

The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP:co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT+ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe2+ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe2+. We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.

FIG. 1.

The corrinoid adenosylation pathway in S. enterica. Fpr, NADP⁺:ferredoxin (flavodoxin) reductase; FAD/FADH₂, flavin adenine dinucleotide/reduced flavin adenine dinucleotide; FMNH₂, reduced flavin mononucleotide; FMNH · , flavin mononucleotide semiquinone; PPP_i, inorganic triphosphate; Ado, 5-deoxyadenosine; FldA, flavodoxin A. The corrin ring is schematized as shown with the Co ion in the appropriate oxidation state.

FIG. 2.

The ethanolamine utilization (eut) operon of S. enterica.

FIG. 3.

In vivo evidence that eutT encodes a cobalamin adenosyltransferase enzyme. Strains were grown in NCE minimal medium supplemented with ethanolamine as a carbon and energy source and CNCbl (200 nM) at 37°C. Cultures of cobA⁺ strains reached stationary phase approximately 22 h after inoculation. VOC, vector-only control; peutT⁺, pEUT7.

FIG. 4.

Corrinoid adenosyltransferase activity is detectable in cobA cell lysates. Strains JE7204 (cobA eutT/pBAD24) and JE7205 (cobA eutT/pEUT7) were grown on ethanolamine as the sole C source in minimal medium supplemented with AdoCbl. Cells were harvested and lysed anoxically with 0.2 M Tris Cl (pH 8.0) in 1× BugBuster reagent (Novagen) containing protease inhibitor. Protein content was normalized with fresh buffer. The KBH₄ corrinoid adenosylation procedure was followed (35), except that the amount of reductant was reduced to 3 μmol per reaction mixture. Reactions were allowed to proceed for 1 h at 37°C, and the AdoCbl concentration was determined by photolysis. Lysate containing EutT by overexpression from the pEUT7 plasmid produced AdoCbl at levels dependent on the amount of protein added per reaction mixture. Only a background level decrease in absorbance was detected from the vector-only negative control. Each data point represents the average of at least three reactions.

FIG. 5.

EutT adenosyltransferase activity is sensitive to air and metal chelation. Substrates and protein were added, and the reaction was initiated by a shift to 37°C. EutT-dialyzed extract was prepared as described in Materials and Methods. ATP, 500 μM; CobA, 40 μg of protein; EutT⁺, 100 μg of protein (EutT-enriched extract); Batho., 10 or 100 μM bathophenanthroline; air, EutT-enriched extract exposed to air for 20 min and then flushed with O₂-free N₂ gas before addition to the reaction mixture. AdoCbl production was determined by comparing the A₅₂₅ after 30 min with that after 10 min of photolysis. Each data point represents the average of at least two reactions. ND, not detected.