Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii.
D-Ornithine aminomutase from Clostridium 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. The oraS 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 alpha(2)beta(2)-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 K(m) values for d-ornithine, 5'-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5'-phosphate (PLP) are 44.5 +/- 2.8, 0.43 +/- 0.04, and 1.5 +/- 0.1 microm, respectively; the k(cat) is 6.3 +/- 0.1 s(-1). The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, and D-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 recombinant D-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.
Chen
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HP
Biochemistry Department, China Medical College, Taichung 404, Taiwan. hpchen@mail.cmc.edu.tw
Wu
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SH
Lin
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YL
Chen
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CM
Tsay
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D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D003038:Cobamides / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D019751:Intramolecular Transferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D015335:Molecular Probes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009952:Ornithine / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D003038:Cobamides; D008024:Ligands; D015335:Molecular Probes; D011994:Recombinant Proteins; C414173:adensoylcobinamide; D009952:Ornithine; D019751:Intramolecular Transferases; C021913:ornithine 5,4-aminomutase; D008239:Lysine