INACTIVATION OF THE REGULATORY PROTEIN-B OF SOLUBLE METHANE MONOOXYGENASE FROM METHYLOCOCCUS-CAPSULATUS (BATH) BY PROTEOLYSIS CAN BE OVERCOME BY A GLY TO GIN MODIFICATION

The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-lengt h active form and truncated farms, B' and B ''. Electrospray ionisatio n mass spectrometry (ESI-MS) was used to identify a cleavage site betw een Met12 and Gly13, such that 12 amino acids were lost from the N-ter minus of protein B. This truncate was designated B' and molecular mass es were assigned to proteins B and B' of 15852.6 +/- 0.4 Da and 14629. 5 +/- 0.3 Da, respectively. A cleavage site between Gln29 and Val30 wa s also identified such that 29 amino acids were lost from the N-termin us of protein B. This truncate was designated B '' and had a molecular mass of 12709.93 +/- 0.02 Da. Proteins B' and B '' were found to be i nactive in the sMMO system. Addition of protease inhibitors or the het erologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation. Expr ession of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from a coli using affinity chroma tography resulted in preparations of protein B with higher enzyme acti vities than that of wild-type protein B. However, ESI-MS confirmed tha t protein B' was still present. Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations. ES I-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow pro tein B to maintain a certain conformation necessary for enzyme activit y and this may control the activity of sMMO in response to the supply of methane to the cell.