EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of co-substrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a “supernucleophilic” cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g. Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate “base-on” and “base-off” Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of “base-off” Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.

(adenosylcobalamin, AdoCbl, CoB 12 ) is an essential nutrient for animals, lower eukaryotes, and many prokaryotes. The unique organometallic bond of AdoCbl, between the cobalt ion of the corrinoid and the carbon of the 5=-deoxyadenosyl group, lies at the center of its reactivity. The formation of this bond is catalyzed by ATP:cob(I)alamin adenosyltransferase (ACAT) enzymes (1). There are three types of ACAT enzymes, namely, CobA, PduO, and EutT (2-8). They do not share sequence similarity at the nucleotide level or at the amino acid level, making them a good example of convergent evolution. All three ACAT types were discovered in Salmonella enterica, and they serve distinct growth requirements. The CobA type is the housekeeping ACAT, whereas PduO and EutT are specialized enzymes needed for growth on 1,2-propanediol and ethanolamine, respectively (7, 9).Extensive work has been done to understand the mechanism of corrinoid adenosylation performed by the CobA and PduO type ACATs. These two types of enzymes facilitate the thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin by generating a cob(II)alamin four-coordinate intermediate in the active site of the enzyme (10-12). Both CobA and PduO use conserved aromatic side chains located directly below the cobalt ion to displace the lower ligand of Cbl and generate the four-coordinate species (5, 13). The reduction potential of the cobalt ion in this intermediate is raised enough so that free or protein-bound dihydroflavins can reduce cob(II)alamin to cob(I)alamin (5). Unlike the CobA and PduO type ACAT enzymes, nothing is known about the mechanism of how the thermodynamically unfavorable reduction to cob(II)alamin is facilitated when EutT type ACAT enzymes adenosylate Cbl. In fact, the EutT type enzyme is the least understood of the three types of ACATs because it has not been isolated to homogeneity.We previously reported a method for enriching a sample with EutT using detergents (6). We proposed that the EutT type ACAT enzyme contained a metal cofactor because the protein contains an HX 11 CCXXC 83 motif that resembles those found in Fe/S-containing proteins and inferred that the metal cofactor was labile to oxidation and could be extracted from the protein by metal chelators (6). Problems with the isolation of EutT hindered its biochemical characterization.In this study, we successfully purified wild-type EutT to homogeneity in the absence of detergents. Herein, we report the initial kinetic characterization of EutT and show that the enzyme requires a divalent, transition state metal ion and is most active when in complex with ferrous ions. Results from experiments using homogeneous EutT demonstrate that free dihydroflavins can reduce cob(II)alamin, suggesting that EutT, similar to CobA and PduO, facilitates the unfavorable reduction of cob(II)alamin. This is the first known example of an ACAT requiring an inorganic cofactor for activity. MATERIALS AND METHODS Construction of expression vectors.To generate a recombinant construct of EutT with a...

ATP:Co(I)rrinoid adenosyltransferases (ACATs) are enzymes that catalyze the formation of adenosylcobalamin (AdoCbl, coenzyme B12) from cobalamin and ATP. There are three families of ACATs, namely CobA, EutT and PduO. In Salmonella enterica, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment. Here, we report the crystal structure of CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin. This provides the first crystallographic evidence for the existence of cob(II)alamin in the active site of CobA. The structure suggests a mechanism in which the enzyme adopts a closed conformation and two residues, Phe91 and Trp93, displace 5,6-dimethylbenzimidazole (DMB), the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond. In vivo and in vitro mutational analysis of Phe91 and Trp93 emphasize the important role of bulky hydrophobic side chains in the active site. The proposed manner in which CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond appears to be analogous to that utilized by the PduO-type ACATs, where in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains.

CobA from Salmonella enterica (SeCobA) is a member of the family of ATP:Co(I)rrinoid adenosyltransferase (ACAT) enzymes that participate in the biosynthesis of adenosylcobalamin by catalyzing the transfer of the adenosyl group from an ATP molecule to a reactive Co(I)rrinoid species transiently generated in the enzyme active site. This reaction is thermodynamically challenging, as the reduction potential of the Co(II)rrinoid precursor in solution is far more negative than that of available reducing agents in the cell (e.g., flavodoxin), precluding nonenzymic reduction to the Co(I) oxidation state. However, in the active sites of ACATs, the Co(II)/Co(I) redox potential is increased by >250 mV via the formation of a unique four-coordinate (4c) Co(II)rrinoid species. In the case of the SeCobA ACAT, crystallographic and kinetic studies have revealed that the phenylalanine 91 (F91) and tryptophan 93 (W93) residues are critical for in vivo activity, presumably by blocking access to the lower axial ligand site of the Co(II)rrinoid substrate. To further assess the importance of the F91 and W93 residues with respect to enzymatic function, we have characterized various SeCobA active-site variants using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our data provide unprecedented insight into the mechanism by which SeCobA converts the Co(II)rrinoid substrate to 4c species, with the hydrophobicity, size, and ability to participate in offset π-stacking interactions of key active-site residues all being critical for activity. The structural changes that occur upon Co(II)rrinoid binding also appear to be crucial for properly orienting the transiently generated Co(I) “supernucleophile” for rapid reaction with cosubstrate ATP.

Summary Salmonella enterica catabolizes ethanolamine inside a compartment known as the metabolosome. The ethanolamine utilization (eut) operon of this bacterium encodes all functions needed for the assembly and function of this structure. To date, the roles of the EutQ and EutP were not known. Herein we show that both proteins have acetate kinase activity, and that EutQ is required during anoxic growth of S. enterica on ethanolamine and tetrathionate. EutP and EutQ-dependent ATP synthesis occurred when enzymes were incubated with ADP, Mg(II) ions and acetyl-phosphate. EutQ and EutP also synthesized acetyl-phosphate from ATP and acetate. Although EutP had acetate kinase activity, ΔeutP strains lacked discernable phenotypes under the conditions where ΔeutQ strains displayed clear phenotypes. The kinetic parameters indicate that EutP is a faster enzyme than EutQ. Our evidence supports the conclusions that EutQ and EutP represent novel classes of acetate kinases. We propose that EutQ is necessary to drive flux through the pathway under physiological conditions, preventing a buildup of acetaldehyde. We also suggest that ATP generated by these enzymes may be used as a substrate for EutT, the ATP-dependent corrinoid adenosyltransferase, and for the EutA ethanolamine ammonia-lyase reactivase.