| Abstract
| - In Salmonella typhimurium, formation of the cobalt−carbon bond in the biosynthetic pathwayfor adenosylcobalamin is catalyzed by the product of the cobA gene which encodes a protein of 196amino acid residues. This enzyme is an ATP:co(I)rrinoid adenosyltransferase which transfers an adenosylmoiety from MgATP to a broad range of co(I)rrinoid substrates that are believed to include cobinamide,its precursor cobyric acid and probably others as yet unidentified, and hydroxocobalamin. Three X-raystructures of CobA are reported here: its substrate-free form, a complex of CobA with MgATP, and aternary complex of CobA with MgATP and hydroxycobalamin to 2.1, 1.8, and 2.1 Å resolution,respectively. These structures show that the enzyme is a homodimer. In the apo structure, the polypeptidechain extends from Arg28 to Lys181 and consists of an α/β structure built from a six-stranded parallelβ-sheet with strand order 324516. The topology of this fold is very similar to that seen in RecA protein,helicase domain, F1ATPase, and adenosylcobinamide kinase/adenosylcobinamide guanylyltransferase wherea P-loop is located at the end of the first strand. Strikingly, the nucleotide in the MgATP·CobA complexbinds to the P-loop of CobA in the opposite orientation compared to all the other nucleotide hydrolases.That is, the γ-phosphate binds at the location normally occupied by the α-phosphate. The unusual orientationof the nucleotide arises because this enzyme transfers an adenosyl group rather than the γ-phosphate. Inthe ternary complex, the binding site for hydroxycobalamin is located in a shallow bowl-shaped depressionat the C-terminal end of the β-sheet of one subunit; however, the active site is capped by the N-terminalhelix from the symmetry-related subunit that now extends from Gln7 to Ala24. The lower ligand of cobalaminis well-ordered and interacts mostly with the N-terminal helix of the symmetry-related subunit. Interestingly,there are few interactions between the protein and the polar side chains of the corrin ring which accountsfor the broad specificity of this enzyme. The corrin ring is oriented such that the cobalt atom is located∼6.1 Å from C5‘ of the ribose and is beyond the range of nucleophilic attack. This suggests that aconformational change occurs in the ternary complex when Co(III) is reduced to Co(I).
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