Crystal structure of a PduO‐type ATP:cobalamin adenosyltransferase from <i>Burkholderia thailandensis</i>

Abstract

ATP:cobalamin adenosyltransferase (EC 2.5.1.17), an enzyme in the vitamin B12 (cobalamin) metabolic pathway, converts cobalamin to adenosylcobalamin (coenzyme B12) by transferring a 5′-deoxyadenosyl moiety from ATP to the cobalt atom in cobalamin.1 Adenosylcobalamin functions as a radical reservoir, and enzymes utilize this organometallic cofactor to catalyze 1,2-rearrangement reactions that interchange a hydrogen atom and a variable group (OH, NH2, or a carbon-containing group) on adjacent carbons.2 Amino acid sequence analysis have revealed three types of adenosyltransferases: CobA, PduO, and EutT.3 The crystal structure of CobA from Salmonella typhimurium4 showed a homodimer with mixed α/β-folds, but the structure of PduO from Thermoplasma acidophilum5 revealed a homotrimer with mainly α-folds (a five-helix bundle); a three-dimensional structure of EutT has not yet been reported. They share little sequence identity (<20%) and have thus far yielded different three-dimensional structures, even though they catalyze the same reactions, suggesting that particular types of adenosyltransferases are specialized for particular B12-dependent enzymes or for the de novo cobalamin biosynthesis.6 Among the adenosyltransferases, PduO is the most widely distributed enzyme with homologs occurring from archaea to human. PduO has drawn considerable interest, because the human PduO homolog (MMAB gene) is defective in at least two cobalamin metabolic disorders: methylmalonic aciduria and metabolic ketoacidosis.7 Several crystal structures of PduO adenosyltransferases are available in the Protein Data Bank database, but to date only four crystal structures have been reported (from T. acidophilum,5 human,8 Lactobacillus reuteri,9 and Sulfolobus tokodaii10). Of these structures, only two (human and L. reuteri) described the substrate-binding site with ATP bound or detailed the role of the conserved residues around the N-terminus. The PduO protein from Burkholderia thailandensis possesses 35 and 40% sequence identity to the T. acidophilum and human PduO proteins, respectively. It was predicted to be a PduO-type adenosyltransferase, because all conserved residues comprising the putative PduO active site aligned completely with known PduO adenosyltransferases. In this report, we have determined crystal structures for this PduO adenosyltransferase from B. thailandensis at a 1.8-Å resolution, with and without its substrates, a Mg2+ ion and an ATP molecule (MgATP). Furthermore, we demonstrated concrete and specific functions for the conserved ATP-binding residues by comparing the native and MgATP-complexed structures. These structural studies provide valuable information for understanding the molecular mechanism of human methylmalonic aciduria, a cobalamin metabolic disorder disease. The full-length PduO gene was amplified by polymerase chain reaction (PCR) from the genomic DNA of B. thailandensis. The PCR product was digested with NcoI and XhoI restriction enzymes and ligated into the pET-28a expression vector (Novagen), which contained a hexahistidine tag at the C-terminus. The resulting plasmid was transformed into E. coli BL21 (DE3), and the cells were grown at 37°C in Luria-Bertani medium supplemented with kanamycin (50 μg/mL). Expression of the recombinant PduO protein was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the cells had reached an optical density at 600 nm of about 0.45. The cells were grown for an additional 12 h at 18°C and harvested by centrifugation at 6000 rpm for 30 min at 4°C. The pelleted cells were suspended in buffer A (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 10 mM β-mercaptoethanol) and homogenized by sonication. The crude lysate was centrifuged at 15,000 rpm for 1 h at 4°C, and the supernatant was loaded onto a Ni2+-chelated HiTrap metal-chelating column (GE Healthcare) that had been equilibrated with buffer A. The protein was eluted with a linear gradient of buffer A containing 1M imidazole. The fractions