Structural and biochemical studies of the regulation of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

摘要

Methane monooxygenase (MMO) enzymes catalyze the oxidation of methane to methanol in methanotrophic bacteria. Some methanotrophs, including Methylococcus capsulatus (Bath), express two forms of MMO. At high copper levels, a membrane-bound or particulate MMO (pMMO) is expressed whereas a soluble form of MMO (sMMO) is produced when copper is limited. The mechanism of this copper switch is not understood.;This work focuses on understanding the regulation of sMMO because it has great potential in bioremediation applications. When copper availability is minimal, four regulatory proteins, MmoS, MmoQ, MmoG and MmoR, are proposed to be involved in a phosphorylation/interaction scheme whereby MmoS senses the copper concentration and MmoR activates expression of sMMO in M. capsulatus (Bath).;All four regulatory proteins were cloned, expressed and purified as a first step toward understanding their roles in the copper switch mechanism. Though obvious copper binding motifs are not present in MmoS, analysis of its sequence reveals two N-terminal PAS domains that may be the sensors of copper in M. capsulatus (Bath). Biochemical characterization of MmoS revealed the presence of a flavin adenine dinucleotide (FAD) cofactor with a redox potential of E0 = -290 +/- 2 mV at pH 8.0 and 25°C. A new model for the regulation of sMMO was developed based on the biochemical characterization of MmoS, also involving MmoQ, MmoR and MmoG.;The X-ray crystal structure of the M. capsulatus (Bath) MmoS N-terminal PAS domains was determined to 2.34 A resolution. Both domains exhibit the prototypical PAS domain alpha/beta topology and are structurally similar, but the FAD cofactor is housed solely within one PAS domain. The structure suggests key residues that may be involved in MmoS FAD redox chemistry and signal transduction, and provides new insight into the architecture of tandem PAS domains.;Finally, in an effort to increase our knowledge of flavoprotein chemistry, we have crystallized and solved the structure of styrene monooxygenase A from Pseudomonas putida S12 to 2.9 A resolution. Model building is currently in progress. The completed structure will provide insight into the mechanism of SMOA and expand our knowledge of flavoprotein monooxygenase chemistry.