Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

Departments of Molecular Biosciences and of Chemistry, Northwestern University, Evanston, Illinois 60208, United States.
Biochemistry (Impact Factor: 3.02). 11/2011; 50(47):10231-40. DOI: 10.1021/bi200801z
Source: PubMed


Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

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    • "A second metal centre located within the membrane-spanning regions (henceforth referred to as the subunit-C site) was originally modelled as coordinated by ligands from PmoA (Glu195) and PmoC (Asp153, His160 and His173) and contained zinc or copper (Lieberman & Rosenzweig, 2005). Based on the higher resolution Methylocystis strain M structure, an alternate model of this site was proposed involving only PmoC residues (Culpepper & Rosenzweig, 2012; Smith et al., 2011). Alternative models for tricopper and di-iron metal centres have also been proposed by Chan et al. (2007) and Martinho et al. (2007), respectively. "
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    ABSTRACT: The hydrocarbon monooxygenase (HMO) of Mycobacterium NBB4 is a member of the copper-containing membrane monooxygenase (CuMMO) superfamily, which also contains particulate methane monooxygenases (pMMOs) and ammonia monooxygenases (AMOs). CuMMOs have broad applications due to their capacity to attack difficult substrates of environmental and industrial relevance. Most of our understanding of CuMMO biochemistry is based on pMMOs and AMOs as models. All three available structures are from pMMOs. These share two metal sites: a dicopper centre coordinated by histidine residues in subunit-B and a "variable-metal" site coordinated by carboxylate and histidine residues from subunit-C. The exact nature and role of these sites still remain contested. Significant barriers to progress have been the physiologically-specialised nature of methanotrophs and autotrophic ammonia-oxidizers, lack of a recombinant expression system for either enzyme, and difficulty in purification of active protein. In this study we use the newly-developed HMO model system to perform site-directed mutagenesis (SDM) on the predicted metal-binding residues in the HmoB and HmoC of NBB4 HMO. All mutations of predicted HmoC metal centre ligands abolished enzyme activity. Mutation of a predicted copper-binding residue of HmoB (B H155V) reduced activity by 81%. Mutation of a site that shows conservation within physiologically-defined subgroups of CuMMOs was shown to reduce relative HMO activity towards larger alkanes. Our study demonstrates that the modelled dicopper site of subunit-B is not sufficient for HMO activity and that a metal centre predicted to be coordinated by residues in subunit-C is essential for activity.
    Microbiology 03/2014; 160(Pt_6). DOI:10.1099/mic.0.078584-0 · 2.56 Impact Factor
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    • "A methane monooxygenase from Methylocystis sp. strain M has been reported to have a copper-active site that turned it into a powerful copper oxidase [30]. Manganese and iron were also found to enhance the PM102 oxygenase activity to some extent, indicating that Mn/Fe may play a role as an enzyme cofactor in absence of Cu. "
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    ABSTRACT: Stenotrophomonas maltophilia PM102 (NCBI GenBank Acc. no. JQ797560) is capable of growth on trichloroethylene as the sole carbon source. In this paper, we report the purification and characterisation of oxygenase present in the PM102 isolate. Enzyme activity was found to be induced 10.3-fold in presence of 0.7 mM copper with a further increment to 14.96-fold in presence of 0.05 mM NADH. Optimum temperature for oxygenase activity was recorded at 36°C. The reported enzyme was found to have enhanced activity at pH 5 and pH 8, indicating presence of two isoforms. Maximum activity was seen on incubation with benzene compared to other substrates like TCE, chloroform, toluene, hexane, and petroleum benzene. K m and V max for benzene were 3.8 mM and 340 U/mg/min and those for TCE were 2.1 mM and 170 U/mg/min. The crude enzyme was partially purified by ammonium sulphate precipitation followed by dialysis. Zymogram analysis revealed two isoforms in the 70% purified enzyme fraction. The activity stain was more prominent when the native gel was incubated in benzene as substrate in comparison to TCE. Crude enzyme and purified enzyme fractions were assayed for TCE degradation by the Fujiwara test. TCE biotransformation products were analysed by FT-IR spectroscopy.
    09/2013; 2013(7):723680. DOI:10.1155/2013/723680
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    • "Three metal centers were identified per protomer from the crystal structure: the first and second sites are located in pmoB, and the third site is located within the lipid bilayer. The first site contains a single metal ion assigned as copper, while the second metal site is a conserved dinuclear site that contains two copper ions, which was also found in subsequent pMMO structures from Methylosinus trichosporium OB3b (Hakemian et al., 2008) and Methylocystis species Strain M (Smith et al., 2011), respectively. The third metal center, modeled as a single zinc ion, is located within the lipid bilayer; it was proposed to be derived from the crystallization buffer but could be occupied by other metal ions in vivo. "
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    Frontiers in Microbiology 03/2013; 4:58. DOI:10.3389/fmicb.2013.00058 · 3.99 Impact Factor
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