Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath).
Methylococcus capsulatus (Bath) uses a soluble methane monooxygenase (sMMO) to catalyse the oxidation of methane to methanol. sMMO is comprised of three components; A, B and C. Protein C (the reductase) transfers electrons from NADH to protein A (the hydroxylase) which contains the active site, and protein B regulates this electron flow. The five genes encoding the sMMO proteins and their subunits are clustered and have been cloned in Escherichia coli. A DNA fragment containing mmoB, the gene encoding protein B, was subcloned into pT7-5, a plasmid of the T7 RNA polymerase promoter expression system. Upon induction, E. coli expressed protein B which was fully functional after purification. The gene encoding protein C, mmoC, was amplified with unique restriction sites at each end using the polymerase chain reaction and then subcloned into pT7-7 (a plasmid similar to pT7-5 but containing its own ribosome-binding site and ATG start codon). Protein C expressed in E. coli was also found to be functional. This is the first report of the functional expression of methanotroph methane monooxygenase genes in a heterologous host and represents a significant step forward in our analysis of the assembly and catalysis of sMMO.
West
C A
CA
Department of Biological Sciences, University of Warwick, Coventry, UK.
Salmond
G P
GP
Dalton
H
H
Murrell
J C
JC
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D008772:Methylococcaceae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012995:Solubility
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010105:Oxygenases; C021555:methane monooxygenase