The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH:quinone oxidoreductase complex from Methylococcus capsulatus Bath. J Bacteriol 2003 Oct;185(19):5755-64
Date
09/18/2003Pubmed ID
13129946Pubmed Central ID
PMC193963DOI
10.1128/JB.185.19.5755-5764.2003Scopus ID
2-s2.0-0141838951 (requires institutional sign-in at Scopus site) 185 CitationsAbstract
Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol.min(-1).mg of protein(-1). Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 microM) to 95 microM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.
Author List
Choi DW, Kunz RC, Boyd ES, Semrau JD, Antholine WE, Han JI, Zahn JA, Boyd JM, de la Mora AM, DiSpirito AAMESH terms used to index this publication - Major topics in bold
Cell MembraneCopper
Culture Media
Detergents
Gene Expression Regulation, Bacterial
Gene Expression Regulation, Enzymologic
Glucosides
Iron
Methylococcus capsulatus
Multienzyme Complexes
NAD(P)H Dehydrogenase (Quinone)
Oxygenases
