Introduction To Methanotrophs

Methanotrophs are a unique group of Gram-negative methane-oxidizing bacteria that grow aero-bically on methane as their sole carbon and energy source (1). Some methanotrophs can also be grown on methanol; however, they are generally considered to be obligate in their requirement for one-carbon compounds and cannot be grown on other multicarbon substrates. Since 1970, they have received considerable attention from microbiologists and industrialists because they can be exploited in biotransformations and bioremediation processes (2). For example, they can be used to convert propylene to propylene oxide and they can degrade the groundwater pollutant trichloroethylene. They are also used in the production of single-cell protein from methane, a relatively inexpensive feedstock. In addition to their commercial potential, methanotrophs also play a key role in the cycling of methane in the natural environment because they oxidize much of the methane produced in anaerobic sediments such as wetlands, peat bogs, and paddy fields, thereby mitigating release of methane to the atmosphere and reducing global warming. It is also now clear that some methanotrophs in aerobic soils can oxidize atmospheric concentrations of methane (e.g., 1-2 ppm), thereby further reducing global warming as a result of methane. Methane-oxidizing bacteria can be enriched for and isolated from a wide variety of environments, including freshwater, sediments, soils, seawater, peat bogs, tundra, paddy fields, and hot springs.

The methanotrophs form a physiologically and phylogenetically coherent group of bacteria. They can be divided into two groups based on a number of distinct morphological and physiological characteristics. Type I methanotrophs, which include the genera Methylococcus, Methylomicrobium, Methylobacter, Methylosphaera, Methylocaldum, and Methylomonas, are y-proteobacteria that possess bundles of unusual intracytoplasmic membranes found throughout the cell. They are bacteria that possess predominantly 16-carbon fatty acids in their membranes and fix formaldehyde, a central intermediate in the methane oxidation pathway, into cell biomass using the ribulose monophosphate cycle. Type-II methanotrophs, which include the genera Methylosinus, Methylocystis, and Methylocella are a-proteobacteria that have their membranes arranged around the periphery of the cell, contain predominantly 18-carbon fatty acids in their membranes and fix carbon at the level of formaldehyde via the serine cycle.

From: Handbook of Copper Pharmacology and Toxicology Edited by: E. J. Massaro © Humana Press Inc., Totowa, NJ

Fig. 1. Pathway of methane oxidation in methane-oxidizing bacteria. The two forms of methane monooxygenase, pMMO and sMMO, use different electron donors. NADH transfers electrons to the sMMO, but the electron donor for pMMO is not yet known. 2H represents reducing equivalents generated. Carbon is assimilated at the level of formaldehyde and proceeds via the ribulose monophosphate cycle (type-I methanotrophs) or serine cycle (type-II methanotrophs).

Fig. 1. Pathway of methane oxidation in methane-oxidizing bacteria. The two forms of methane monooxygenase, pMMO and sMMO, use different electron donors. NADH transfers electrons to the sMMO, but the electron donor for pMMO is not yet known. 2H represents reducing equivalents generated. Carbon is assimilated at the level of formaldehyde and proceeds via the ribulose monophosphate cycle (type-I methanotrophs) or serine cycle (type-II methanotrophs).

2. METHANE OXIDATION PATHWAY

The pathway for bacterial methane oxidation is shown in Fig. 1. Methane is oxidized by methanotrophs to CO2 via the intermediates methanol, formaldehyde, and formate. Approximately 50% of the formaldehyde arising from the oxidation of methane is assimilated into cell carbon and the remainder is oxidized to CO2 and lost from the cell (3). The dissimilatory reactions, from formaldehyde to CO2, generate reducing power for biosynthesis and for the initial methane-oxidation step. Recently, it has been shown that methanotrophs also have a tetrahydromethanopterin-based C1 metabolism that provides a further route for the dissimilation of formaldehyde. Therefore there are several routes for the removal of the toxic intermediate formaldehyde within the cell. The first and key enzyme in the methane-oxidation pathway is methane monooxygenase (MMO). There are two distinct types of MMO enzymes: a soluble, cytoplasmic enzyme complex (sMMO) and a membrane-bound particulate enzyme system (pMMO). The following sections will outline the properties of these two distinct enzyme systems.

3. BIOCHEMISTRY OF SOLUBLE METHANE MONOOXYGENASE

It was thought until recently that sMMO was only found in methanotrophs of the genera Methylosinus, Methylocystis, and Methylococcus. However, sMMO has subsequently been characterized in some Methylomonas (4) and Methylomicrobium (5) species. However, not all methanotrophs contain this enzyme and it is not expressed in, for example, Methylocaldum and Methylobacter species. sMMO is only expressed when the copper-to-biomass ratio of the culture is low (i.e., under "low-copper" growth conditions). There is also evidence that copper can inhibit the activity of the sMMO reductase component (6). The sMMO enzyme has an extremely broad substrate specificity, co-oxidizing a wide range of alkanes, alkenes, substituted aliphatic compounds, and even aromatic compounds such as naphthalene, making it an extremely attractive enzyme for biotransformation processes and bioremediation (2).

The first sMMO enzymes to be examined in detail were those from the type-I methanotroph Methylococcus capsulatus (Bath) and the type-II methanotroph Methylosinus trichosporium OB3b (7-9). They are probably now the most well characterized of these enzymes. sMMO is a member of a family of nonheme iron-containing enzyme complex consisting of three components: hydroxylase,

Table 1

Some Members of the Family of Nonheme Binuclear Iron-Containing Oxygenases

Oxygenase

Microorganism

Methane monooxygenase (sMMO) Methane monooxygenase (sMMO) Methane monooxygenase (sMMO) Methane monooxygenase (sMMO) Alkene monooxygenase Isoprene monooxygenase Alkene monooxygenase Benzene monooxygenase Toluene-o-xylene monooxygenase Toluene-3-monooxygenase Phenol hydroxylase Catechol dioxygenase Toluene-4-monooxygenase Dimethyl sulfide oxygenase Alkane hydroxylase

Methylosinus trichosporium Methylococcus capsulatus Methylocystis sp. Strain M Methylomonas sp. KSWIII Nocardia corallina Rhodococcus sp. AD45 Xanthobacter sp. Py2 Pseudomonas aeruginosa Pseudomonas stutzeri Ralstonia pickettii Ralstonia sp. E2 Pseudomonas putida Pseudomonas mendocina Acinetobacter sp. Pseudomonas oleovorans protein B, and protein C. The hydroxylase component (also known as protein A) has three subunits, a, p, and y of approx 60, 45, and 20 kDa, respectively, which are arranged in an a2p2y2 configuration. The a-subunit contains a nonheme bis-^-hydroxo-bridged binuclear iron center at the active site of the enzyme, where methanol is formed from methane and oxygen. The crystal structure of hydroxylase components from M. capsulatus (Bath) and M. trichosporium OB3b have been reported (10,11). The di-iron center of the hydroxylase resides below the "floor" of two canyon regions formed by its a- and P-subunits. Like some other multicomponent oxygenases such as phenol hydroxylase and toluene monooxygenase, sMMO contains a small regulatory or coupling protein, protein B, the activity and stability of which appears to be controlled by proteolysis at its amino terminus (12). The structures of protein B from M. capsulatus (Bath) and M. trichosporium OB3b have been determined by nuclear magnetic resonance (NMR), and provide insights into its interaction with the hydroxylase (13,14). Protein B at low concentrations converts the hydroxylase from an oxidase to a hydroxylase and stabilizes intermediates needed for oxygen activation. Higher, saturating amounts of protein B dramatically increases the rates of formation of intermediates and improves catalysis of methane to methanol by sMMO. The third component, called protein C, is a 39-kDa NADH-dependent [2Fe-2S]- and FAD-containing reductase, which accepts electrons from NADH2 and transfers them to the di-iron site of the hydroxylase. sMMO is a member of the family of nonheme binuclear iron proteins that includes hemerythrin, ribonucleotide reductase, and purple acid phosphatase and that, together with several other oxygenases, appears to form a subclass of C-H activating oxygenases. Some representatives of this family of di-iron proteins are given in Table 1.

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