Cob(I)yrinic acid a,c-diamide adenosyltransferase
The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) to Co(II) by a one-electron transfer. (ii) Co(II) is reduced to Co(I) in a second single-electron transfe, and (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP to leave the cobalt atom in a Co(III) state. The enzyme responsible for the adenosylation reaction is the product of the gene cobO in the aerobic bacterium Pseudomonas denitrificans and of the gene cobA in the anaerobic bacterium Salmonella typhimurium. This entry represents the cobA gene product, which shows a preference for ATP and Mn(II), although it is able to transfer a variety of nucleosides to the cobalt, including CTP, UTP and GTP, in decreasing order of preference and to use Mg(ii) instead of Mn(II).
Reference Protein and Structure
- Sequence
-
P31570
(2.5.1.17)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (Bacteria)
- PDB
-
1g64
- THE THREE-DIMENSIONAL STRUCTURE OF ATP:CORRINOID ADENOSYLTRANSFERASE FROM SALMONELLA TYPHIMURIUM. COBALAMIN/ATP TERNARY COMPLEX
(2.1 Å)
- Catalytic CATH Domains
-
3.40.50.300
(see all for 1g64)
- Cofactors
- Magnesium(2+) (1)
Enzyme Reaction (EC:2.5.1.17)
Enzyme Mechanism
Introduction
The mechanism involves the one-electron reduction of a Co(II)rrinoid precursor to form a “supernucleophilic” Co(I) species, which performs a nucleophilic attack on the 5′-carbon of ATP to yield the adenosylated product. The reaction proceeds via complexation of the enzyme active site with cosubstrate ATP. This promotes the binding of the Co(II)Cbl and its conversion to a 4c species via removal of the axial ligand. One-electron reduction of this species produces a Co(I)Cbl intermediate that is properly oriented for nucleophilic attack on the 5′-carbon of ATP to form AdoCbl and triphosphate.
The nucleotide in the MgATP·CobA complex binds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases. That is, the gamma-phosphate binds at the location normally occupied by the alpha-phosphate. The unusual orientation of the nucleotide arises because this enzyme transfers an adenosyl group rather than the gamma-phosphate.
Catalytic Residues Roles
| UniProt | PDB* (1g64) | ||
| Glu128 | Glu128A | Forms part of the Mg(II) binding site. | metal ligand |
| Thr42 | Thr42A | Forms part of the Mg(II) binding site, also involved in stabilising the negative charge in the active site. | metal ligand, electrostatic stabiliser |
| Trp93, Phe91 | Trp93A, Phe91A | Phe91 and Trp93 play a critical role in CobA function; might be stabilising cobalamin via pi-sigma interactions; hydrophobic side chains at positions 91 and 93 play a role in the conversion of five- to four-coordinate cob(II)alamin to allow the formation of the cob(I)alamin nucleophile. | electrostatic stabiliser |
| Lys39, Asn37, Thr43, Lys41, Arg51 | Lys39A, Asn37A, Thr43A, Lys41A, Arg51B | N37, K39, K41, T42 involved in binding triphosphate group. The extra negative charge is neutralised by the inclusion of Arg51B (from the 2-fold related subunit) and Thr43 in the coordination sphere of the gamma-phosphate. | electrostatic stabiliser |
Chemical Components
References
- Pallares IG et al. (2014), Biochemistry, 53, 7969-7982. Spectroscopic studies of the Salmonella enterica adenosyltransferase enzyme SeCobA: molecular-level insight into the mechanism of substrate Cob(II)alamin activation. DOI:10.1021/bi5011877. PMID:25423616.
- Moore TC et al. (2012), Biochemistry, 51, 9647-9657. Structural insights into the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme: critical role of residues Phe91 and Trp93. DOI:10.1021/bi301378d. PMID:23148601.
- Buan NR et al. (2006), J Bacteriol, 188, 3543-3550. Studies of the CobA-type ATP:Co(I)rrinoid adenosyltransferase enzyme of Methanosarcina mazei strain Go1. DOI:10.1128/JB.188.10.3543-3550.2006. PMID:16672609.
- Stich TA et al. (2005), J Am Chem Soc, 127, 8710-8719. Spectroscopic and computational studies of the ATP:corrinoid adenosyltransferase (CobA) from Salmonella enterica: insights into the mechanism of adenosylcobalamin biosynthesis. DOI:10.1021/ja042142p. PMID:15954777.
- Buan NR et al. (2005), J Biol Chem, 280, 40948-40956. 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. DOI:10.1074/jbc.M506713200. PMID:16207720.
- Fonseca MV et al. (2002), J Biol Chem, 277, 33127-33131. The ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2'-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct. DOI:10.1074/jbc.M203893200. PMID:12080060.
- Bauer CB et al. (2001), Biochemistry, 40, 361-374. Three-Dimensional Structure of ATP:Corrinoid Adenosyltransferase fromSalmonella typhimuriumin Its Free State, Complexed with MgATP, or Complexed with Hydroxycobalamin and MgATP†,‡. DOI:10.1021/bi002145o. PMID:11148030.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Lys41A | electrostatic stabiliser |
| Thr43A | electrostatic stabiliser |
| Asn37A | electrostatic stabiliser |
| Lys39A | electrostatic stabiliser |
| Thr42A | electrostatic stabiliser |
| Arg51B | electrostatic stabiliser |
| Thr42A | metal ligand |
| Glu128A | metal ligand |
| Phe91A | electrostatic stabiliser |
| Trp93A | electrostatic stabiliser |