Direct evidence for a soluble methane monooxygenase from type I methanotrophic bacteria: purification and properties of a soluble methane monooxygenase from Methylomonas sp. GYJ3.
The hydroxylase and reductase components of a soluble methane monooxygenase from type I methanotrophs--Methylomonas sp. GYJ3--were purified by a multiple-step LC procedure. The hydroxylase (approximately 240 kDa, determined by an HPLC-size exclusion chromatography method) has three subunits with molecular masses of 56, 43, and 27 kDa, suggesting that the enzyme has an (alphabeta gamma)2 subunit structure. The HPLC method was developed to purify the hydroxylase component, and the purified protein has a specific activity of 541 nmol propene oxide x mg(-1) protein x min(-1), which is two times the specific activity of the protein purified by the two-step LC procedure. The iron content in the hydroxylase purified by the two-step LC procedure is 2.1 mol of Fe per mole of protein, but the iron content in the protein by the HPLC procedure is 3.78 mol of Fe per mole of protein. The diversity of iron contents in this protein is due mainly to the use of different purification methods. The reductase has a molecular mass of 42 kDa. The UV-VIS spectrum of the protein is similar to that of proteins from other methanotrophs, suggesting that the protein contains a FAD cofactor and a [2Fe-2S] center. The partially purified component B stimulated the MMO activity of the hydroxylase and reductase system by 40-fold.
Shen
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State Key Laboratory of Oxo-synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, People's Republic of China. runnan@ns.lzb.ac.cn
Yu
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Ma
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Li
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D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007501:Iron / Q000032:analysis; D008667:Metalloproteins; D008697:Methane / Q000378:metabolism; D008772:Methylococcaceae / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification*; D011487:Protein Conformation; D012995:Solubility; D013053:Spectrophotometry
D008667:Metalloproteins; D007501:Iron; D010105:Oxygenases; C021555:methane monooxygenase; D008697:Methane