Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-lysine 5, 6-aminomutase from Clostridium sticklandii.
D-Lysine 5,6-aminomutase from Clostridium sticklandii catalyzes the 1,2-shift of the epsilon-amino group of D-lysine and reverse migration of C5(H). The two genes encoding 5,6-aminomutase have been cloned, sequenced, and expressed in Escherchia coli. They are adjacent on the Clostridial chromosome and encode polypeptides of 57. 3 and 29.2 kilodaltons. The predicted amino acid sequence includes a conserved base-off 5'-deoxyadenosylcobalamin binding motif and a 3-cysteine cluster in the small subunit, as well as a P-loop sequence in the large subunit. Activity of the recombinant enzyme exceeds that of the 5,6-aminomutase purified from C. sticklandii by 6-fold, presumably due to the absence of bound, inactive corrinoids in the recombinant enzyme. The K(m) values for adenosylcobalamin and pyridoxal 5'-phosphate are 6.6 and 1.0 microM, respectively. ATP does not have a regulatory effect on the recombinant protein. The rapid turnover associated inactivation reported for the enzyme purified from Clostridium is also seen with the recombinant form. Aminomutase activity does not depend on structural or catalytic metal ions. Electron paramagnetic resonance experiments with [(15)N-dimethylbenz-imidazole]adenosylcobalamin demonstrate base-off binding, consistent with other B(12)-dependent enzymes that break unactivated C-H bonds.
Chang
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Institute for Enzyme Research, Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53705, USA.
Frey
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics*; D003038:Cobamides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D019751:Intramolecular Transferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000032:analysis
D003038:Cobamides; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D019751:Intramolecular Transferases; C403167:D-lysine 5,6-aminomutase; C000913:cobamamide; D015032:Zinc