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 Fe 2؉ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe 2؉ . 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.The biosynthesis of AdoCbl (coenzyme B 12 ) is unique to some prokaryotes. Multiple functions (encoded by Ͼ25 genes) are required for the assembly of this complex molecule (36). The chief structural feature of AdoCbl is the presence of an adenosyl moiety liganded to the Co ion of cobalamin via a covalent Co-C bond. In the enterobacterium Salmonella enterica, corrinoid adenosylation is required for de novo synthesis and for the assimilation of incomplete precursors such as cobinamide (12). Insights into the corrinoid adenosylation pathway in S. enterica ( Fig. 1) were recently reported, including the three-dimensional crystal structure of the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme responsible for the formation of the Co-C bond (2,(13)(14)(15)35). The cobA gene in S. enterica is not part of the 17-gene cob operon (27, 33) and appears to be constitutively expressed (34). In this bacterium, CobA is the housekeeping adenosyltransferase needed for de novo biosynthesis and for the assimilation of exogenous corrinoids. However, CobA is not the only corrinoid adenosyltransferase present in S. enterica. Other large operons like the 1,2-propanediol utilization (pdu) and ethanolamine utilization (eut) operons appear to encode their own corrinoid adenosyltransferases. Johnson et al. recently reported evidence that the pduO gene of S. enterica encodes an ATP:cobalamin adenosyltransferase (19). The eutT gene of the eut operon was suggested to encode the corrinoid adenosyltransferase enzyme for this pathway, but support for this assignment was inconclusive (20). Interestingly, CobA, PduO, and EutT do not have an ancestor in common, suggesting ...

Computer-assisted Docking of Flavodoxin with the ATP:Co(I)rrinoid Adenosyltransferase (CobA) Enzyme Reveals Residues Critical for Protein-Protein Interactions but Not for Catalysis

“…Hence, the metE cobA strain is an AdoCbi, Cbl, or methionine auxotroph. When grown on ethanolamine as the carbon and energy source, AdoCbl synthesized by CobA is needed to activate transcription of the ethanolamine utilization (eut) operon and to provide AdoCbl for ethanolamine ammonia-lyase function (18,32).…”

In Salmonella enterica, Ethanolamine Utilization Is Repressed by 1,2-Propanediol To Prevent Detrimental Mixing of Components of Two Different Bacterial Microcompartments

“…Prior studies showed that transcription of the eut operon of S. enterica is induced by the combination of ethanolamine and Ado-B 12 (35,36). Here, we looked at the effects of 1,2-propanediol (1,2-PD) on the induction of the eut operon using two lacZ reporter genes ( Table 2).…”

“…In Salmonella, induction of the eut operon requires both ethanolamine and coenzyme B 12 (Ado-B 12 ), and this dual-effector regulation is mediated by the positive activator protein EutR (34). The eutR gene is transcribed from its own constitutive promoter and also by the main eut operon promoter, which is activated by ethanolamine and Ado-B 12 (35,36) (Fig. 1.).…”

Diverse Bacterial Microcompartment Organelles

“…The eutA gene encodes an EAL reactivase (162), and the eutT gene encodes a corrinoid cobalamin adenosyltransferase that converts cob(I)alamin to Ado-B 12 , the required cofactor of EAL (163)(164)(165). The genes used for the metabolism of acetaldehyde include eutE, which encodes an aldehyde dehydrogenase that converts acetaldehyde plus HS-CoA plus NAD ϩ to acetyl-CoA plus NADH (7,161).…”