ABSTRACT
The epoxide hydrolase (EH) from Corynebacterium sp. C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic-interaction chromatography. The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32,140 Da. The gene encoding Corynebacterium EH was located on a 3.5-kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N-terminal and an internal amino acid sequence. Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 [Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. (1997) J. Biol. Chem. 272, 14650- 14657), particularly around the catalytic site. All of these proteins belong to the alpha/beta-hydrolase-fold family of enzymes. Similarity to the mammalian microsomal EH is weaker.
Subject(s)
Corynebacterium/enzymology , Epoxide Hydrolases/isolation & purification , Amino Acid Sequence , Animals , Base Composition , Base Sequence , Binding Sites , Cloning, Molecular , Codon/genetics , Corynebacterium/genetics , Corynebacterium/growth & development , Cyclohexanes/metabolism , Cyclohexenes , DNA Primers/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Epoxide Hydrolases/chemistry , Epoxide Hydrolases/genetics , Genes, Bacterial , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.
Subject(s)
Methylococcaceae/enzymology , Oxygenases/metabolism , Electrochemistry , Electron Transport , Flavin-Adenine Dinucleotide/metabolism , Iron-Sulfur Proteins/metabolism , NAD/metabolism , Oxidation-Reduction , Protein Binding , Solubility , SpectrophotometryABSTRACT
A major improvement in the purification of the oxygenase protein (component A) of the methane monooxygenase has been effected. By employing high-pressure gel permeation chromatography several purification steps may be omitted from the previously published scheme. Furthermore the yield of the protein is enhanced and more importantly the recovered protein displays an increased specific activity, unlike that purified by other techniques.
Subject(s)
Bacterial Proteins/isolation & purification , Escherichia coli Proteins , Methylococcaceae/enzymology , Oxygenases/isolation & purification , Chromatography, Gel , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Iron/analysis , SolubilityABSTRACT
Methylococcus capsulatus (Bath) possesses a multi-component methane monooxygenase which catalyzes in vivo the conversion of methane to methanol. Component A of this enzyme system, believed to be the oxygenase component, has been purified to near homogeneity (95%). The native protein has a molecular weight of approximately 210,000 and is comprised of three subunits of Mr = 54,000, 42,000, and 17,000, which appear to be present in stoichiometric amounts suggesting an alpha 2, beta 2, gamma 2 arrangement in the native protein. Purified preparations of the protein are virtually colorless and examination of the uv/visible absorption spectrum revealed a peak around 280-290 nm and thereafter a steady decrease in absorbance to longer wavelengths. The ESR spectrum of the oxidized protein gave a signal at g = 4.3, presumably due to rhombic iron, and a radical signal at g = 2.01. Upon reduction with dithionite, a signal at g = 1.934 appeared. Chemical analyses of our purified preparations revealed the presence of iron (2.3 mol/mol) and zinc (0.2-0.5 mol/mol): molybdenum, copper, nickel, heme, and acid-labile sulfur were all virtually absent. On ultra thin layer isoelectric focusing, purified component A was judged to have a pI between 5.0 and 5.1. Extracts prepared from a variety of other methanotrophs failed to show any cross-reaction to antibody raised against M. capsulatus component A.
