The regulatory protein and component interactions of soluble methane monooxygenase

• Main Author: Callaghan, Anastasia J.
• Published: University of Warwick 2000
• Subjects: 572; QH301 Biology
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364628

The purpose of this study was to investigate the regulatory protein (protein B) and component interactions of soluble methane monooxygenase (sMMO). sMMO is a multi-component enzyme which catalyses the oxidation of methane to methanol. It consists of three proteins, a hydroxylase, a reductase and protein B (Colby and Dalton, 1978). Protein B contains no metals, cofactors or prosthetic groups and has a molecular mass of 16 kDa. It has been shown that protein B is absolutely necessary for the hydroxylase activity of the sMMO complex and is a powerful regulator of the enzyme (Green and Dalton, 1985). It has also been found that 12 amino acids are cleaved from the N-terminus of protein B from Me. eapsulatus (Bath) to form an inactive truncate, known as protein B' and mutation of the Met12_Gly13 cleavage site to Met12 -GIn I3 to give the single mutant protein G13Q, improved the stability of the protein (Lloyd et al., 1997). Much of this work has concentrated on the study of the catalytic and regulatory significance of the 12 amino acids cleaved from protein B. Mc. eapsulatus (Bath) protein B appears to- cleave autocatalytically, generating the inactive protein B' truncate. The secondary structures of proteins B and B' were seen to be the same, although the overall structure was identified as differing slightly and protein B was shown to be capable of existing in a monomer-dimer equilibrium, whereas protein B' was identified as existing in a monomer form. An homologous protein B from Ms. trichosporium OB3b, identified as being more a-helical in character, has been shown to be more stable than Mc. eapsulatus (Bath) protein B but still undergoes the inactivating cleavage reaction to form truncates, although the cleavage sites differ between the two proteins. The construction, expression and purification of N-terminal truncates of Mc. capsulatus (Bath) protein B identified that the presence of the first 7 amino acids was essential for protein B activity within the sMMO system and a decrease in specific activity was observed as each amino acid from 1 to 7 was lost. Upon loss of the 7th amino acid, tyrosine, the truncate protein was observed to be totally inactive and also much more prone to cleavage, but unchanged in terms of secondary structure. Protein concentration was observed as having an effect on the stability of Mc. capsulatus (Bath) protein B and, the single mutant G13Q, with increased concentrations improving stability. This effect was not observed for the double mutant MI2A:G13Q, although it was shown to be more stable than the other proteins under more dilute conditions. The addition of a magnesium salt also improved the stability of protein B. Studies into the interactions of protein B with the other proteins within the sMMO complex have also been performed. Evidence that the hydroxylase undergoes a large conformational change upon the binding of the reductase and protein B has been obtained and modelled to suggest that one trimer of the hydroxylase dimer rotates by 1800 relative to the other upon complex formation. It also showed the sMMO complex to form in a stoichiometry of 1:2:2 hydroxylase:reductase:protein B. Other data suggest that -sMMO component binding occurs on only one trimer of the hydroxylase dimer under different conditions.