Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii

D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversib le rearrangement of D-ornithine to (2R,4S)-2,4-diaminopentanoic acid. The t wo genes encoding D-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli. The oraS gene, which encodes a protein of 12 1 amino acid residues with M-r 12,800, is situated upstream of the oraE gen e, which encodes a protein of 753 amino acid residues with M-r 82,900. The holoenzyme appears to comprise a alpha (2)beta (2)-heterotetramer. OraS sho ws no significant homology to other proteins in the Swiss-Prot data base. T he deduced amino acid sequence of OraE includes a conserved base-off/histid ine-on cobalamin-binding motif, DXHXXG. OraE was expressed in E. coli as in clusion bodies. Refolding experiments on OraE indicate that the interaction s between OraS and OraE and the binding of either pyridoxal phosphate or ad enosylcobalamin play important roles in refolding process. The K-m values f or D-ornithine, 5'-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5'-phosph ate (PLP) are 44.5 +/- 2.8, 0.43 +/- 0.04, and 1.5 +/- 0.1 muM, respectivel y; the k(cat) is 6.3 +/- 0.1 s(-1). The reaction was absolutely dependent u pon OraE, OraS, AdoCbl, PLP, and D-ornithine being present in the assay; no other cofactors were required. A red-shift in UV-visible absorption spectr um is observed when free adenosylcobinamide is bound by recombinant D-ornit hine aminomutase and no significant change in spectrum when free adenosylco binamide is bound by mutant OraE-H618G, demonstrating that the enzyme binds adenosylcobalamin in base-off/histidine-on mode.