The Three-Dimensional Structures of Nicotinate Mononucleotide:5,6-Dimethylbenzimidazole Phosphoribosyltransferase (CobT) from Salmonella typhimurium Complexed with 5,6-Dimethybenzimidazole and Its Reaction Products Determined to 1.9 Å Resolution^,

Abstract

Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin. Two X-ray structures of CobT are reported here at 1.9 A resolution. First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate. CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit. Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate. These studies show that CobT is a dimer where each subunit consists of two domains. The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold. The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle. The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known. The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit. DMB binds in a hydrophobic pocket created in part by the neighboring small domain. This is consistent with the broad specificity of this enzyme for aromatic substrates [Trzebiatowski, J. R., Escalante-Semerena (1997) J. Biol. Chem. 272, 17662-17667]. The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction. The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands. Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold.

Conversion of Cobinamide into Adenosylcobamide in Bacteria and Archaea

“…The phosphoribosyltransferase activity of CobT has been studied in P. denitrificans (the protein is known as CobU in this bacterium) and serovar Typhimurium (14,58,59). Structural analyses of the CobT protein in its apo form and bound to substrates provided insights into the mechanism of catalysis and the specificity of the enzyme for its base substrate (15)(16)(17). The ADP-ribosyltransferase activity of CobT was recently described, uncovering the previously unknown intermediate of the pathway known as ␣-DAD (␣-5,6-dimethylbenzimidazole adenine dinucleotide) (37).…”

Characterization of Determinants of Ligand Binding to the Nicotinic Acid Receptor GPR109A (HM74A/PUMA-G)

“…From analyses of crystal structures of nicotinic acid bound at diverse prokaryotic proteins [such as nicotinate mononucleotide dimethylbenzimidazole phosphoribosyltransferase (PDB code 1D0V) (Cheong et al, 1999), dihydropteridine reductase (PDB code 1ICR) (Lovering et al, 2001), nicotinate nucleotide dimethylbenzimidazole phosphoribosyltransferase (PDB code 1JHA) (Cheong et al, 2001), dihydrodipicolinate reductase (PDB code 1DRV) (Reddy et al, 1996), and the plant protein ferric soybean leghemoglobin (PDB code 1FSL) (Ellis et al, 1997)], it is evident that the pyridine ring system of the ligand is always bound near aromatic side chains of the protein. Following the structural homology paradigm, we assumed that the binding pocket of GPR109A is also coated by aromatic side chain(s) as an additional binding partner for nicotinic acid.…”

Two distinct pools of B12analogs reveal community interdependencies in the ocean

“…Rhodobacters have the O 2 -dependent corrin ring and DMB pathways (11), whereas Thaumarchaeota likely possess the O 2 -independent pathway for the corrin ring and the O 2 -dependent DMB pathway (10). In some bacteria, pseudocobalamin can be produced if DMB synthesis is impaired; this is due to the natural presence of adenine in cells and enzyme substrate promiscuity that allows adenine to substitute for DMB in some organisms' CobT (22)(23)(24)(25)(26)(27). Cyanobacteria use the O 2 -independent pathway for corrin ring synthesis and neither pathway for DMB synthesis (11,18) (Fig.…”