Article

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: 3.01). 11/2011; 50(47):10231-40. DOI: 10.1021/bi200801z
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Joshua Telser, Aug 09, 2015
1 Follower
 · 
162 Views
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 · 2.84 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps, and compare typical enzymes from various classes with regard to their three-dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyzes, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments.
    Frontiers in Microbiology 03/2013; 4:58. DOI:10.3389/fmicb.2013.00058 · 3.94 Impact Factor
  • Source
    • "Although the first possibility cannot be discarded, it is very unlikely. The pMMO of M. oxyfera shows a high sequence identity to the well-studied enzymes from other organisms with known crystal structures, including amino acids implicated in catalysis (Balasubramanian and Rosenzweig, 2007; Smith et al., 2011). With NO as the direct oxidizing agent, at least some modifications would be expected. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nitric oxide (NO) and nitrous oxide (N(2)O) are among nature's most powerful electron acceptors. In recent years it became clear that microorganisms can take advantage of the oxidizing power of these compounds to degrade aliphatic and aromatic hydrocarbons. For two unrelated bacterial species, the "NC10" phylum bacterium "Candidatus Methylomirabilis oxyfera" and the γ-proteobacterial strain HdN1 it has been suggested that under anoxic conditions with nitrate and/or nitrite, monooxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide only. Similarly, "aerobic" pathways for hydrocarbon degradation are employed by (per)chlorate-reducing bacteria, which are known to produce oxygen from chlorite [Formula: see text]. In the anaerobic methanotroph M. oxyfera, which lacks identifiable enzymes for nitrogen formation, substrate activation in the presence of nitrite was directly associated with both oxygen and nitrogen formation. These findings strongly argue for the role of NO, or an oxygen species derived from it, in the activation reaction of methane. Although oxygen generation elegantly explains the utilization of "aerobic" pathways under anoxic conditions, the underlying mechanism is still elusive. In this perspective, we review the current knowledge about intra-aerobic pathways, their potential presence in other organisms, and identify candidate enzymes related to quinol-dependent NO reductases (qNORs) that might be involved in the formation of oxygen.
    Frontiers in Microbiology 10/2012; 3:273. DOI:10.3389/fmicb.2012.00273 · 3.94 Impact Factor
Show more