Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme.
The genes encoding both components, MutE and MutS, of adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum have been over-expressed in Escherichia coli. This has allowed MutE to be obtained in homogeneous form, free of inhibiting cobamides and traces of MutS. MutE binds MutS cooperatively, with a Hill coefficient of 1.3. The recombinant enzyme has an unchanged Km for L-glutamate, but a much higher specific activity than those previously reported for preparations from clostridia. The apparent Km for adenosylcobalamin was dependent upon the concentration of MutS and varied between 18 microM with equimolar concentrations of MutS and MutE and 5.8 microM with a 5-fold molar excess of MutS over MutE present in the assay. The dissociation constant for adenosylcobalamin was measured directly using equilibrium gel filtration. In the presence of equimolar amounts of MutE and MutS, the apparent Kd was 5.4 microM, but this decreased to 1.8 microM when MutS was present at a 5-fold molar excess. No binding of adenosylcobalamin to MutE was observed in the absence of MutS. This suggests that the (minimal) function for MutS, whose role in the reaction has been unclear until now, is to form part of the adenosylcobalamin-binding site. It seems likely that MutS is representative of a cobalamin-binding domain conserved across several cobalamin-dependent enzymes.
Holloway
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Department of Biochemistry, University of Cambridge, United Kingdom.
Marsh
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D000591:Amino Acid Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D003038:Cobamides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019751:Intramolecular Transferases; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides
D003038:Cobamides; D009838:Oligodeoxyribonucleotides; D000591:Amino Acid Isomerases; D019751:Intramolecular Transferases; C021570:methylaspartate mutase; C000913:cobamamide