New Enzyme Uses Copper to Catalyze Halogenation Reactions

A research team led by Yi Tang, who holds the Ralph M. Parsons Foundation Chair in Chemical Engineering at the UCLA Samueli School of Engineering, has discovered a new type of metal-containing enzyme from a natural fungus that can perform surprising halogenation reactions on organic molecules. 

Published in Nature, the study represents a four-year effort by chemical and biomolecular engineering graduate student Chen-Yu “Yorick” Chiang and research scientist Masao Ohashi. 

In addition to Tang, who is a professor of chemical and biomolecular engineering and bioengineering at UCLA Samueli, the research team also comprises multiple professors in the Department of Chemistry and Biochemistry where Tang holds a faculty appointment, including Joseph Loo, Ken Houk and Jose Rodriguez. Additional collaborators are Yisong “Alex” Guo at Carnegie Mellon University and Shabnam Hematian from the University of North Carolina at Greensboro.

The halogenation reaction, which involves the replacement of a carbon-hydrogen bond with a carbon-halogen bond in organic molecules, is an important synthetic transformation —particularly in the pharmaceutical industry. Adding a functional group of halogen substituents to molecules can dramatically improve their physical and pharmacological properties, as well as providing a reaction handle that can enable further modification. While many synthetic halogenation reagents have been developed, selectively introducing a halogen at the least reactive position as this enzyme does is remarkably difficult.

Halogenases — enzymes that replace carbon-hydrogen bonds with carbon-halogen bonds — have evolved in nature, but the known enzyme family uses iron as a reactive center to achieve halogenation. The reaction scope is therefore limited to substrates that can coordinate with the iron. The new enzyme class uses copper, which will greatly expand the scope of enzymatic halogenations.

The researchers focused on halogen-containing molecules produced by microorganisms in order to find new halogenases. The team identified atpenin A5, a potent antifungal molecule which has two carbon-chlorine bonds at inactivated carbon atoms. By identifying enzymes in the biosynthetic pathway, a protein previously labeled “domain of unknown function (DUF) 3328” was implicated in the halogenation reaction. The authors used AlphaFold, an artificial-intelligence-based protein structure prediction tool, to identify a potential metal binding site that is different from known enzymes. Different characterization techniques, including mass spectrometry, electron paramagnetic resonance and biochemical assays showed that the enzyme binds two copper atoms to catalyze the halogenation reaction with high efficiency. The use of a copper center in an enzyme to perform halogenation is unprecedented in nature.

Utilizing the discovery of the copper-dependent enzyme, Tang’s lab has developed enzymatic halogenation reactions that were not possible with the iron-based halogenases. For example, the enzyme is able to perform iodination of an inactivated carbon-hydrogen bond that has not been demonstrated by enzymes. This is due to the compatibility in electrical properties between copper and iodide, which do not apply to iron. Capitalizing on the unique property of the enzyme’s copper center, other useful functional groups such as thiocyanate and selenocyanate could also be selectively incorporated into the substrate molecule.

“This discovery showed new enzymes with unprecedented properties and catalytic powers can be discovered from nature,” Tang said. “These enzymes perform reactions that are highly challenging by synthetic methods, and can therefore be useful tools for modifying or constructing molecules.”

Further research has since shown such copper-dependent enzymes are widely found in nature, and mostly catalyze unknown reactions. Tang’s research team is now exploring functions of these previously cryptic enzymes to understand their potentials as biocatalysts.

Additional authors on the paper are graduate students Jessie Le and Qingyang Zhou, postdoctoral scholars Panpan Chen and Undramaa Bat-Erdene, and Songrong Qu, an undergraduate student who graduated in 2024. This work was funded by the National Institutes of Health and the U.S. Department of Energy.