Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependent D-Ornithine Aminomutase from Clostridium sticklandii

Abstract d-Ornithine aminomutase fromClostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. TheoraS gene, which encodes a protein of 121 amino acid residues with M r 12,800, is situated upstream of the oraE gene, which encodes a protein of 753 amino acid residues with M r 82,900. The holoenzyme appears to comprise a α2β2-heterotetramer. OraS shows no significant homology to other proteins in the Swiss-Prot data base. The deduced amino acid sequence of OraE includes a conserved base-off/histidine-on cobalamin-binding motif, DXHXXG. OraE was expressed in E. coli as inclusion bodies. Refolding experiments on OraE indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate or adenosylcobalamin play important roles in refolding process. The Km values ford-ornithine, 5′-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5′-phosphate (PLP) are 44.5 ± 2.8, 0.43 ± 0.04, and 1.5 ± 0.1 μm, respectively; thek cat is 6.3 ± 0.1 s−1. The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, andd-ornithine being present in the assay; no other cofactors were required. A red-shift in UV-visible absorption spectrum is observed when free adenosylcobinamide is bound by recombinantd-ornithine aminomutase and no significant change in spectrum when free adenosylcobinamide is bound by mutant OraE-H618G, demonstrating that the enzyme binds adenosylcobalamin in base-off/histidine-on mode.