The University of Texas at Austin Professor Yi Lu and his Welch Catalyst Grant team have an ambitious goal: turning an abundant and free nitrogen source directly into useful chemicals and pharmaceuticals. Today, with the help of a Catalyst Grant, the team is making progress with this high-risk, high-reward research.
Nitrogen gas (N2), which makes up approximately 78 percent of air, is present in most classes of biomolecules, natural products and synthesized compounds. Plants convert atmospheric nitrogen to more useful compounds through nitrogenase (N2ase), but scientists have yet to successfully adapt this enzymatic process to the laboratory. In the early 1900s, researchers developed the Haber-Bosch process to convert nitrogen into ammonia, which is primarily used in fertilizer production. While instrumental in dramatically improving plant yields, the process uses 1 to 2 percent of global energy and creates about 1.4 percent of the world’s carbon dioxide (CO2) emissions.
Dr. Lu’s team aims to not only make ammonia production more sustainable by carrying out this reaction at ambient temperature and pressure, but to take this challenge a step further, moving directly from N2 to high-value chemical products. The plan is to take N2 from air and transfer one or both nitrogen atoms to organic substrates by designing a new class of enzymes called artificial dinitrogen transferases (ArtN2Tases).
While there are numerous enzymes and chemical catalysts capable of activating oxygen (O2) and directly transferring its O to organic substrates or biomolecules, to date there is no enzyme or catalyst capable of a direct N2 transfer reaction (DN2TR). This program has the potential to transform the chemistry of “nitrogenation” by developing a new paradigm for N2 activation and DN2TR. This would allow for the biocatalytic production of a wide variety of important chemical compounds.
“Despite its enormous potential, the program is high-risk as it is much more difficult to activate N2 and capture/transfer its intermediates than commensurate processes for O2,” explained Dr. Lu.
He pointed to major barriers to engineering naturally occurring N2ases. Their complex overall structure and elegant active site, a so-called “metallocofactor” containing up to eight iron sites, complicate mutagenesis and heterologous expression in E. coli or strains that might be used in bioprocessing. There are chemical barriers, as well. It is very difficult to synthesize small molecule catalysts that replicate the essential chemical environment around the metallocluster.
Dr. Lu further clarifies that tightly controlling and tuning these primary and secondary coordination spheres is essential to putting highly reactive intermediates – such as hydrazine (N2H4 ) and diazene (N2H2) – to good use in high-value chemical reactions.
Dr. Lu says he was fortunate to assemble a team of “top notch” Texas researchers with expertise in the five disparate fields needed to tackle the problem:
- Metalloprotein design/engineering (Dr. Lu)
- Synthetic organometallic chemistry (Michael J. Rose, associate professor, UT Austin)
- Organic chemistry of nitrogen transfer reactions (Kami Hull, associate professor, UT Austin)
- Computational chemistry to understand reaction mechanisms (Thomas R. Cundari, Regents Professor of Chemistry, University of North Texas) and
- Synthetic biology to optimize enzymes using machine learning and directed evolution (Andrew Ellington, Kathry M. Fraser Endowed Research Professor of Biochemistry, UT Austin).
This specialized and collaborative team is leveraging its range of complementary expertise: Dr. Lu is working with Dr. Rose to design ArtN2Tases that bind synthetic metal clusters for N2 reduction; Dr. Hull is identifying reactions and designing strategies that will promote reactions between organic substrates and the transient N2 reduction intermediates; and Dr. Ellington is using a new generation of non-canonical amino acids and directed evolution to improve the nascent ArtN2Tases. In parallel, there are efforts to improve the fundamental chemistry of nitrogen reduction. Drs. Lu and Rose will perform structural, spectroscopic and mechanistic studies of ArtN2Tases — guided by Dr. Cundari’s computations — and work with Dr. Hull to capture the intermediates for DN2TRs.
“This unparalleled expertise and our close collaboration allow us to exploit recent advances in chemical/biochemical methods, making this the right time and the right team to tackle this major challenge at the leading edge of chemical catalysis and biocatalysis,” Dr. Lu said.
The research team is taking a novel approach to designing ArtN2Tases – drastically different from both current engineered N2ases and synthetic models – using small, robust proteins free of other subunits or metallocofactors and whose mutants are readily constructed, expressed and purified in E. coli.
“Given nitrogen’s strong bonds and nitrogenase’s complex structures, chemistry and reaction mechanisms, this is a challenge no single group could do,” Dr. Lu added. “It needs a whole team with a wide range of expertise. We will be revolutionizing basic knowledge in chemistry and biology as capturing N2 intermediates from the nitrogenase reaction has never been done before. This research is way out there, and we are so excited that Welch believed in us and had the vision to fund it.”