Missing links in biological methane and ammonia oxidation

Project: Research project

Project Details

Description

Methanotrophic bacteria oxidize methane to methanol in the first step of their metabolic pathway. Whereas current catalysts that can selectively activate the 105 kcal mol-1 C-H bond in methane require high temperatures and pressures, methanotrophs perform this chemistry under ambient conditions using methane monooxygenase (MMO) enzymes. The membrane-bound MMO (pMMO), which utilizes a copper active site, is the predominant methane oxidation catalyst in nature; some methanotrophs can also produce a soluble iron-containing enzyme (sMMO). MMOs have received much attention for their potential applications in the development of new biological gas-to-liquids processes. The only other enzyme known to oxidize methane is ammonia monooxygenase (AMO), a pMMO homolog that converts ammonia to hydroxylamine in nitrifying bacteria. Despite extensive efforts, work on pMMO and AMO has been hindered for the past 30 years by reduced enzymatic activity upon isolation from the native organisms.
To efficiently oxidize methane or ammonia, several basic components are necessary: a properly assembled metal active site, a redox cofactor, an electron transfer pathway from this cofactor to the active site, and a proton transfer pathway to the active site. It is not know how the pMMO active site is assembled in vivo and whether it is properly preserved in vitro. In addition, the identity of the redox cofactor and the means of delivering electrons to pMMO remain unclear. It is quite likely that the persistent issues with pMMO (and AMO) activity are likely related to insufficient knowledge regarding these and other aspects of catalysis. In particular, there could be unidentified protein components or other “missing links” that facilitate loading, assembly, and stabilization of the active site and/or delivery of reductants and protons.
A prime source of these missing links might lie within the operons encoding pMMO and AMO. These operons include the three genes encoding the enzyme subunits, pmoB (or amoB), pmoA (or amoA), and pmoC (or amoC). Directly adjacent to pmoB/amoB is a gene denoted pmoD/amoD followed by three genes encoding putative copper transport proteins, copC, copD, and DUF461. These four genes are coregulated with pMMO genes in a copper-dependent fashion. CopC and DUF461 belong to periplasmic copper chaperone families, and CopD is a putative copper importer. By contrast, PmoD does not belong to any known protein family and close homologues thereof are only found in methane and ammonia oxidizing bacteria.
The objectives of this project are to biochemically and functionally characterize these proteins, focusing on the pMMO operon, and to determine their roles in biological methane oxidation. The experimental plan combines genetic and biochemical approaches with spectroscopy, X-ray crystallography, and advanced biophysical tools. The resultant data may provide new insight into pMMO active site chemistry and redox reactions as well as into the assembly of the larger biological methane oxidation system. The deployment of pMMO and/or methanotrophs in new bioconversion processes requires improved energy efficiencies, carbon yields, and reaction rates. While such advances are potentially attainable through expression of pMMO in heterologous hosts, engineering of methanotrophs, optimization of pMMO, and development of biomimetic catalysts, success will require a full understanding of pMMO function. The elucidation of new protein components that affect pMMO activity would impact such efforts dramatically.
StatusActive
Effective start/end date9/1/168/31/22

Funding

  • Department of Energy (DE-SC0016284 0004)

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