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Verrucomicrobial methanotrophs grow on diverse C3 compounds and use a homolog of particulate methane monooxygenase to oxidize acetone.

ABSTRACT: Short-chain alkanes (SCA; C2-C4) emitted from geological sources contribute to photochemical pollution and ozone production in the atmosphere. Microorganisms that oxidize SCA and thereby mitigate their release from geothermal environments have rarely been studied. In this study, propane-oxidizing cultures could not be grown from acidic geothermal samples by enrichment on propane alone, but instead required methane addition, indicating that propane was co-oxidized by methanotrophs. "Methylacidiphilum" isolates from these enrichments did not grow on propane as a sole energy source but unexpectedly did grow on C3 compounds such as 2-propanol, acetone, and acetol. A gene cluster encoding the pathway of 2-propanol oxidation to pyruvate via acetol was upregulated during growth on 2-propanol. Surprisingly, this cluster included one of three genomic operons (pmoCAB3) encoding particulate methane monooxygenase (PMO), and several physiological tests indicated that the encoded PMO3 enzyme mediates the oxidation of acetone to acetol. Acetone-grown resting cells oxidized acetone and butanone but not methane or propane, implicating a strict substrate specificity of PMO3 to ketones instead of alkanes. Another PMO-encoding operon, pmoCAB2, was induced only in methane-grown cells, and the encoded PMO2 could be responsible for co-metabolic oxidation of propane to 2-propanol. In nature, propane probably serves primarily as a supplemental growth substrate for these bacteria when growing on methane.

SUBMITTER: Awala SI

PROVIDER: S-EPMC8630023 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Particulate methane monooxygenase genes in methanotrophs.

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Project description:The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane-bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N-terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability.

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Effects of zinc on particulate methane monooxygenase activity and structure.

Project description:Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Zinc is a known inhibitor of pMMO, but the details of zinc binding and the mechanism of inhibition are not understood. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. The 2.6 Å resolution crystal structure of Methylocystis species strain Rockwell pMMO reveals two previously undetected bound lipids, and metal soaking experiments identify likely locations for the two zinc inhibition sites. The first is the crystallographic zinc site in the pmoC subunit, and zinc binding here leads to the ordering of 10 previously unobserved residues. A second zinc site is present on the cytoplasmic side of the pmoC subunit. Parallels between these results and zinc inhibition studies of several respiratory complexes suggest that zinc might inhibit proton transfer in pMMO.

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The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b.

Project description:Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

| S-EPMC2664655 | biostudies-literature

Evidence for oxygen binding at the active site of particulate methane monooxygenase.

Project description:Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an ?(3)?(3)?(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the ?-?(2):?(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site.

| S-EPMC3348976 | biostudies-literature

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