ADENOSYLCOBALAMIN-DEPENDENT GLUTAMATE MUTASE FROM CLOSTRIDIUM-TETANOMORPHYUM - OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

The genes encoding both components, MutE and MutS, of adenosylcobalami n-dependent glutamate mutase from Clostridium tetanomorphum have been overexpressed in Escherichia coli. This has allowed MutE to be obtaine d in homogeneous form, free of inhibiting cobamides and traces of MutS . MutE binds MutS cooperatively, with a Hill coefficient of 1.3. The r ecombinant enzyme has an unchanged K-m for L-glutamate, but a much hig her specific activity than those previously reported for preparations from clostridia. The apparent K-m for adenosylcobalamin was dependent upon the concentration of MutS and varied between 18 mu M with equimol ar concentrations of MutS and MutE and 5.8 mu M with a 5-fold molar ex cess of MutS over MutE present in the assay. The dissociation constant for adenosylcobalamin was measured directly using equilibrium gel fil tration. In the presence of equimolar amounts of MutE and MutS, the ap parent K-d was 5.4 mu M but this decreased to 1.8 mu M when MutS was p resent at a 5-fold molar excess, No binding of adenosylcobalamin to Mu tE was observed in the absence of MutS. This suggests that the (minima l) function for MutS, whose role in the reaction has been unclear unti l now, is to form part of the adenosylcobalamin-binding site. It seems likely that MutS is representative of a cobalamin-binding domain cons erved across several cobalamin-dependent enzymes.