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

Publisher Website
Google Scholar
Abstract
The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full‐length active form and truncated forms, B′ and B″. Electrospray ionisation mass spectrometry (ESI‐MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N‐terminus of protein B. This truncate was designated B′ and molecular masses were assigned to proteins B and B′ of 15 852.6 ± 0.4 Da and 14629.5 ± 0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N‐terminus of protein B. This truncate was designated B″ and had a molecular mass of 12 709.93 ± 0.02 Da. Proteins B′ and B″ were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon‐deficient or ompT‐deficient Escherichia coli, did not inhibit B′ formation. Expression of protein B as a glutathione S‐transferase fusion protein and subsequent purification of protein B from E. coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild‐type protein B. However, ESI‐MS confirmed that protein B′ was still present. Alteration of the Met12‐Gly13 cleavage site to Met12‐Gln13 revealed that the stability of G13Q at 20°C and 37°C was higher than that of wild‐type preparations. ESI‐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 protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell.