The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) within the corrinoid to Co(II) by a one-electron transfer. This can occur non-enzymically in the presence of dihydroflavin nucleotides or reduced flavoproteins . (ii) Co(II) is bound by corrinoid adenosyltransferase, resulting in displacement of the lower axial ligand by an aromatic residue. The reduction potential of the 4-coordinate Co(II) intermediate is raised by ~250 mV compared with the free compound, bringing it to within physiological range. This is followed by a second single-electron transfer from either free dihydroflavins or the reduced flavin cofactor of flavoproteins, resulting in reduction to Co(I) . (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP, resulting in transfer of the deoxyadenosyl group and oxidation of the cobalt atom to Co(III) state. Three types of corrinoid adenosyltransferases, not related by sequence, have been described. In the anaerobic bacterium Salmonella enterica they are encoded by the cobA gene (a housekeeping enzyme involved in both the de novo biosynthesis and the salvage of adenosylcobalamin), the pduO gene (involved in (S)-propane-1,2-diol utilization), and the eutT gene (involved in ethanolamine utilization). The first two types, which produce triphosphate, are classified as EC 2.5.1.17, corrinoid adenosyltransferase, while the EutT type hydrolyses triphosphate to diphosphate and phosphate during catalysis and is thus classified separately.
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The enzyme appears in viruses and cellular organisms
The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) within the corrinoid to Co(II) by a one-electron transfer. This can occur non-enzymically in the presence of dihydroflavin nucleotides or reduced flavoproteins [1]. (ii) Co(II) is bound by corrinoid adenosyltransferase, resulting in displacement of the lower axial ligand by an aromatic residue. The reduction potential of the 4-coordinate Co(II) intermediate is raised by ~250 mV compared with the free compound, bringing it to within physiological range. This is followed by a second single-electron transfer from either free dihydroflavins or the reduced flavin cofactor of flavoproteins, resulting in reduction to Co(I) [4]. (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP, resulting in transfer of the deoxyadenosyl group and oxidation of the cobalt atom to Co(III) state. Three types of corrinoid adenosyltransferases, not related by sequence, have been described. In the anaerobic bacterium Salmonella enterica they are encoded by the cobA gene (a housekeeping enzyme involved in both the de novo biosynthesis and the salvage of adenosylcobalamin), the pduO gene (involved in (S)-propane-1,2-diol utilization), and the eutT gene (involved in ethanolamine utilization). The first two types, which produce triphosphate, are classified as EC 2.5.1.17, corrinoid adenosyltransferase, while the EutT type hydrolyses triphosphate to diphosphate and phosphate during catalysis and is thus classified separately.
interaction of Co(II)cobalamin and cob(II)inamide (Co(II)Cbi+) with EutT in the absence and presence of cosubstrate ATP. EutT displays a similar substrate specificity as Salmonella enterica EutT and can bind both Co(II)cobalamin and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)cobalamin to a four-coordinate species. Listeria monocytogenes EutT achieves a more than 98% conversion yield of the five-coordinate to four-coordinate state on addition of just over one molar equivalent of cosubstrate MgATP
the reduced catalytic activity of EutT wild-type in complex with Co relative to the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of four-coordinate Co(II)Cbl. The Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate cob(II)alamin intermediate. Residue Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site
the reduced catalytic activity of EutT wild-type in complex with Co relative to the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of four-coordinate Co(II)Cbl. The Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate cob(II)alamin intermediate. Residue Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site
adenosyltransferase enzymes lower the thermodynamic barrier of the Co2+ to Co+ reduction needed for the formation of the organometalic Co-C bond of adenosylcobalamin. Cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes
EutT converts cob(II)alamin to an effectively four-coordinate Co(II) species so as to facilitate Co(II) to Co(I) reduction. The mechanism for bond dissociation involves binding of the nucleotide loop of Co(II)Cbl to a site remote from the Co(II) center. EutT fails to promote axial ligand dissociation for the substrate analogue cob(II)inamide, a natural precursor of cob(II)alamin. EutT can adenosylate complete and incomplete Co(I)rrinoids
during the synthesis of adenosylated corrinoids, reduction of co(II)rrinoids occurs on the adenosyltransferase enzyme once it has complexed with its substrate and ATP
in Salmonlella typhimurium, three types of corrinoid adenosyltransferases have been described. CoBa is a housekeeping enzyme involved in both the de novo biosynthesis and the salvage of adenosylcobalamin. The PduO adenosyl transferase is encoded in an operon (pdu) for cobalamin-dependent propanediol degradation and is induced by propanediol. Transferase EutT is encoded within the operon for ethanolamine utilization (eut). CobA produces sufficient Ado-B12 to initiate eut operon induction and to serve as a cofactor for EA-lyase when B12 levels are high. Once the eut operon is induced, the EutT transferase supplies more Ado-B12 during the period of high demand
lack of corrinoid adenosyltransferase CobA, Ec 2.5.1.17, and EutT blocks ethanolamine utilization. EutT is necessary and sufficient for growth of an Salmonella typhimurium CobA EutT mutant strain on ethanolamine as a carbon and energy or nitrogen source. Cell extracts enriched for EutT protein contain strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity is only detected in extracts maintained under anoxic conditions
replacement affects binding of the cobalamin substrate to the active site, mutant strain fails to synthesize enough adenosylcobalamin to support growth
replacement affects binding of the cobalamin substrate to the active site, mutant strain fails to synthesize enough adenosylcobalamin to support growth
replacement affects binding of the cobalamin substrate to the active site, mutant strain fails to synthesize enough adenosylcobalamin to support growth
replacement affects binding of the cobalamin substrate to the active site, mutant strain fails to synthesize enough adenosylcobalamin to support growth
mutation in the HX11CCX2C(83) motif required for the tight binding of the divalent metal, substitution causes a large decrease in the affinity of EutT for Co(II)
mutation in the HX11CCX2C(83) motif required for the tight binding of the divalent metal, large negative effect on enzyme activity, only capable of very slow turnover
mutation in EutT to corresponding residues in Lactobacillus reuteri PduO, Ec 2.5.1.17, associated with ATP binding and formation of an intersubunit salt bridge. The equivalent residues in EutT enzyme affect ATP binding
mutation in EutT to corresponding residues in Lactobacillus reuteri PduO, Ec 2.5.1.17, associated with ATP binding and formation of an intersubunit salt bridge. The equivalent residues in EutT enzyme affect ATP binding
Spectroscopic study of the EutT adenosyltransferase from Listeria monocytogenes evidence for the formation of a four-coordinate cob(II)alamin intermediate
Park, K.; Mera, P.E.; Moore, T.C.; Escalante-Semerena, J.C.; Brunold, T.C.
Unprecedented mechanism employed by the Salmonella enterica EutT ATP Co(I)rrinoid adenosyltransferase precludes adenosylation of incomplete co(II)rrinoids
Spectroscopic studies of the EutT adenosyltransferase from Salmonella enterica Evidence of a tetrahedrally coordinated divalent transition metal cofactor with cysteine ligation