A multidisciplinary study identifies the active site of Nature’s chosen enzyme for conversion of methane to methanol
A team of researchers at Northwestern University have recently identified the active site in pMMO where methane is converted to methanol, a major stride in learning how methanotrophs accomplish their remarkable chemistry. These findings are published in the journal Nature Catalysis.
Methane is a powerful greenhouse gas, produced in large quantities by a variety of human activities, with approximately 84 times the heat-trapping ability of carbon dioxide. A major goal of modern chemistry is to develop ways of capturing methane and converting it to something more useful, like methanol, a valuable chemical, and biofuel. Although human chemists struggle to accomplish this difficult reaction, Nature contains many microbial chemists, known as methanotrophic bacteria, that are able to convert methane to methanol with ease.
Methanotrophs are bacteria that consume methane gas as their sole source of carbon and of energy. Although methane cannot be used as a food source by most organisms, methanotrophs are able to convert methane gas to liquid methanol because they possess specialized enzymes known as methane monooxygenases (MMOs) that carry out this reaction.
The predominant MMO enzyme is known as particulate methane monooxygenase (pMMO). Despite intense investigation, relatively little had been learned about how pMMO catalyzes the difficult transformation of methane to methanol.
pMMO is difficult to study for several reasons. The methanotrophs that produce pMMO can be finicky, requiring growth in large-scale fermentors under a constant flow of methane gas. pMMO is a membrane enzyme, meaning it is embedded in the membranes of these bacteria, making the enzyme difficult to purify and investigate. Finally, pMMO is a copper enzyme, meaning it uses copper to convert methane to methanol, but there are several copper centers in pMMO, which poses the question: which of the multiple copper centers is the active site where methane is converted to methanol.
A collaborative research team from the labs of Brian Hoffman and Amy Rosenzweig (the senior authors of the paper) used a multidisciplinary approach, combining multiple techniques to overcome the challenges intrinsic to studying pMMO. Hoffman is the Charles E. and Emma H. Morrison Professor of Chemistry in Northwestern’s department of Chemistry. Rosenzweig is the Weinberg Family Distinguished Professor of Life Sciences in Northwestern’s department of chemistry.
The team probed the active site of the enzyme through use of product analog molecules, molecules that resemble methanol (the natural product of pMMO) to. The co-first authors of the paper, Frank Tucci (a PhD candidate in the Rosenzweig lab) and Richard Jodts (a former jointly-advised student from the Hoffman and Rosenzweig labs who recently earned his PhD) decided to first place the pMMO enzymes in native lipid nanodiscs, tiny membrane particles that form a stabilizing belt around pMMO, overcoming the challenges intrinsic to working with a membrane enzyme.
They then added the product analog molecules to samples of pMMO stabilized in nanodiscs, causing the product analog molecules to bind specifically at the pMMO active site. They first detected the binding of the product analog in the pMMO active site using a powerful spectroscopic technique known as electron-nuclear double resonance (ENDOR) spectroscopy, a specialty of the Hoffman lab. ENDOR allowed the team to specifically detect the product analog interacting with the copper of the pMMO active site
Next, the team used a cutting-edge technique known as cryoelectron microscopy (cryoEM), which has recently become a method of choice in the Rosenzweig lab for studying pMMO. CryoEM allows scientists to combine many 2D images of individual large molecules, like pMMO, to determine the 3D structure of said molecule. The team was able to use cryoEM to visualize high-resolution 3D structures of pMMO that showed the product analog molecule binding in the active site of pMMO.
Remarkably, the ENDOR results and the cryoEM results were in beautiful agreement, both showing in geometric detail how the product analog molecule binds to the copper active site of pMMO.
“This is a spectacular example of combining different techniques and expertises to address fundamental molecular questions, and provides a new foundation for our future work on methane-oxidizing enzymes,” says Rosenzweig.
This landmark study represents several firsts. This study was the first in which cryoEM and ENDOR were combined to fully characterize a metal active site. ENDOR identifies and characterizes the metal to which the analogue binds, and gives details of their interaction, but cannot place that active site within the protein; cryoEN is not able to specifically identify metals, but could identify where the analogue binds, and thus place it within the enzyme structure. The study thereby answered the longstanding, and surprisingly challenging, fundamental, question about pMMO: where is the active site where pMMO converts methane to methanol?
“Indeed, the combination of these techniques introduces a new strategy that likely will be used in characterizing a broad range of enzymes,” says Brian Hoffman.
With the answer to this question, scientists can now study pMMO function in much greater detail and begin the work of engineering and synthesizing catalysts that mimic the ability of pMMO to convert methane to methanol.
Despite these important findings, many interesting questions about pMMO remain: How does methane access the active site? How is methanol ejected from the active site? Which factors are most important for pMMO activity? How do other proteins interact with pMMO?
Next, the Rosenzweig lab team hopes to address these questions using cutting-edge methods to image pMMO directly in its native membrane, or even whole methanotroph cells, while ENDOR can interrogate intracellular enzyme sites, just as it does the nanodisk-bound enzyme. This approach would allow the researchers to zoom in on pMMO, taking snapshots of the enzyme it in its native environment.