Methanotrophic bacteria consume 30 million metric tons of methane per year and have captivated researchers for their natural ability to convert the potent greenhouse gas into usable fuel. Yet we know very little about how the complex reaction occurs, and this limits our ability to use the double benefit to our advantage. By studying the enzyme—particulate methane monooxygenase (pMMO)—that the bacteria use to catalyze the reaction, a team at Northwestern University has now discovered key structures that may drive the process, and suggest that their findings could ultimately lead to the development of human-made biological catalysts that convert methane gas into methanol.

“If we don’t understand exactly how the enzyme performs this difficult chemistry, we’re not going to be able to engineer and optimize it for biotechnological applications,” said Northwestern’s Amy Rosenzweig, PhD, senior author of the team’s published paper in Science. “Methane has a very strong bond, so it’s pretty remarkable there’s an enzyme that can do this … If you want to optimize the enzyme to plug it into biomanufacturing pathways or to consume pollutants other than methane, then we need to know what it looks like in its native environment and where the methane binds … You could use bacteria with an engineered enzyme to harvest methane from fracking sites or to clean up oil spills.”

Rosenzweig and colleagues described their study and results in a paper titled “Recovery of particulate methane monooxygenase structure and activity in a lipid bilayer.” Rosenzweig is the Weinberg Family Distinguished Professor of Life Sciences in Northwestern’s Weinberg College of Arts and Sciences, where she holds appointments in both molecular biosciences and chemistry.

Scientists have engineered methanotrophs to produce a range of products, but low yields and poor conversion efficiencies mean the processes aren’t economically viable, the team explained. “For methane bioconversion to be transformative, the initial step—oxidation of methane to methanol—must be optimized, which requires molecular-level understanding of the main enzyme responsible, particulate methane monooxygenase (pMMO).” “The enzyme comprises PmoA (β), PmoB (α), and PmoC (γ), arranged as a trimer of αβγ protomers,” the investigators further noted. “The crystal structures of detergent-solubilized pMMO from multiple methanotrophic species have revealed the presence of three copper-binding sites.”

Cryo-EM illuminated never-before-seen structures in the membrane of the protein. [Credit to Northwestern University]

However, pMMO, a copper-dependent enzyme, is a particularly difficult protein to study because it’s embedded in the bacterial cell membrane. Typically, when researchers study methanotrophic bacteria they use a harsh process in which the proteins are ripped out of the cell membranes using a detergent solution. While this procedure effectively isolates the enzyme, it also kills all enzyme activity and limits how much information researchers can gather—like monitoring a heart without the heartbeat. “pMMO activity decreases upon solubilization in detergent, and purified samples exhibit zero methane oxidation activity, which means that the structures do not represent the active enzyme,” the scientists commented. “Crystal structures determined using inactive, detergent-solubilized pMMO lack several conserved regions neighboring the proposed active site.”

In particular, the team explained, about 25 residues within the PmoC subunit are lost in electron density maps for any pMMO crystal structure. These residues, which correspond to the most highly conserved part of the PmoC sequence, are predicted to reside adjacent to a key copper binding site. “The ambiguity in this region has precluded the identification of any potential cavities for methane and oxygen binding,” the team stated. “It is likely that both of these limitations—the loss of activity and the disorder in PmoC—are attributable to the removal of pMMO from its native membrane environment.” The team reasoned that disruption of the lipid bilayer, as well as detergent solubilization and purification steps may lead to conformational changes and so the separation of any bound metal and/or lipid cofactors.

For the newly reported study the team used an entirely new technique to study pMMO. First author Christopher Koo, a PhD candidate in Rosenzweig’s lab, wondered if by putting the enzyme back into a membrane that resembles its native environment, they could learn something new. Koo used lipids from the bacteria to form a membrane within a protective particle called a nanodisc, and then embedded the enzyme into that membrane.

The researchers used cryo-electron microscopy (cryo-EM), a technique that is well suited to studying membrane proteins because the lipid membrane environment is undisturbed throughout the experiment. Their approach allowed them to visualize the atomic structure of the active enzyme at high resolution for the first time. “By recreating the enzyme’s native environment within the nanodisc, we were able to restore activity to the enzyme,” Koo said. “Then, we were able to use structural techniques to determine at the atomic level how the lipid bilayer restored activity. In doing so, we discovered the full arrangement of the copper site in the enzyme where methane oxidation likely occurs.”

Rosenzweig added, “As a consequence of the recent ‘resolution revolution’ in cryo-EM, we were able to see the structure in atomic detail. What we saw completely changed the way we were thinking about the active site of this enzyme.” The authors further explained, “Multiple nanodisc-embedded pMMO structures determined by cryo–electron microscopy to 2.14- to 2.46-angstrom resolution reveal the structure of pMMO in a lipid environment. The resulting model includes stabilizing lipids, regions of the PmoA and PmoC subunits not observed in prior structures, and a previously undetected copper-binding site in the PmoC subunit with an adjacent hydrophobic cavity.

“These first structures of active pMMO, obtained by embedding the enzyme in a native lipid bilayer, provide critical insight into pMMO structure and function,” the authors further pointed out. “This substantially revised view of pMMO, obtained after >15 years of crystallographic characterization, underscores the importance of studying membrane proteins in their native environments and the potential of high-resolution cryo-EM combined with membrane mimetic technology.”

Rosenzweig said that the cryo-EM structures provide a new starting point to answer the questions that continue to pile on. How does methane travel to the enzyme active site? Or the methanol travel out of the enzyme? How does the copper in the active site do the chemical reaction? “These structures provide a revised framework for understanding and engineering pMMO function,” the authors stated.

Next, the team plans to study the enzyme directly within the bacterial cell using a forefront imaging technique called cryo-electron tomography (cryo-ET). If successful, the researchers will be able to see exactly how the enzyme is arranged in the cell membrane, determine how it operates in its truly native environment and learn whether other proteins around the enzyme interact with it. These discoveries would provide a key missing link to engineers.

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