Illustration of enzymes

Copper-Dependent Enzyme Catalyzes Unprecedented Reactions

By Yi Tang Email Yi Tang

Penny Jennings
Heidi Opdyke

A team of chemists from the University of California, Los Angeles, has discovered a new type of metal-containing enzyme from a natural fungus that can perform surprising halogenation reactions on organic molecules. The study, published in Nature, demonstrates, for the first time, that a copper-dependent enzyme can perform halogenation.

“This discovery showed that new enzymes with unprecedented properties and catalytic powers can be discovered from Nature,” said Yi Tang, the Parsons Family Foundation Professor in Chemical and Biomolecular Engineering at UCLA. “These enzymes perform reactions that are highly challenging by synthetic methods and can therefore be useful tools for modifying or constructing molecules.” 

Carnegie Mellon University’s Yisong (Alex) Guo, whose research focuses on metal-containing enzymes, used electron paramagnetic resonance spectroscopy to confirm that the newly discovered enzyme contains two copper atoms at its active center.

The use of a copper center in an enzyme to perform halogenation is unprecedented in nature.

In organic molecules, the halogenation reaction replaces a carbon-hydrogen bond with a carbon-halogen bond. It is a highly important synthetic transformation, particularly in the pharmaceutical industry. Functionalization of molecules with halogen substituents (fluorine, chlorine, bromine or iodine) can dramatically improve their physical and pharmacological properties, as well as providing a reaction handle that can enable further modification of the molecules. While many synthetic halogenation reagents have been developed, achieving selectivity, particularly introducing halogen at the least reactive position as this enzyme does, is remarkably difficult.

Nature has evolved enzymes called halogenases that perform halogenation reactions. Until now, the only enzyme family known to do these types of reactions involved enzymes with an iron center. 

“Currently the prevalent idea is that if you want to perform C-H activation and a halogenation reaction, you should look to using an iron-based enzyme. My colleagues at UCLA have made a fundamental discovery that can potentially enrich our chemical toolbox with copper-based enzymes,” said Guo, an associate professor in Carnegie Mellon’s Department of Chemistry. “This is the first discovery that a copper enzyme can even do such a challenging C-H activation reaction and specifically install a chloride into a specific carbon position.”

Graduate student Chen-Yu (Yorick) Chiang and research scientist Masao Ohashi, both in UCLA’s Department of Chemical and Biomolecular Engineering, led the effort to look for new halogenases by focusing on halogenated molecules produced by microorganisms. They identified the molecule atpenin A5, a highly potent antifungal compound that featured two carbon-chlorine bonds at unactivated carbon atoms. They identified enzymes in the biosynthetic pathway and implicated a protein previously labeled as “domain of unknown function (DUF) 3328” as being key to the halogenation reaction. They then worked with Associate Professor Jose Rodriguez to use the AlphaFold protein structure prediction tool to identify a potential metal binding site that is different from known enzymes. 

The team used multiple characterization techniques, including biochemical assays and mass spectrometry, to show that the DUF enzyme binds two copper atoms to catalyze the challenging halogenation reaction with high efficiency. They reached out to Guo for additional confirmation of the enzyme’s potential structure at its metal center.

In his research Guo uses different spectroscopy techniques, including Mössbauer, synchrotron radiation-based spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy, to characterize and understand the metal center in metalloenzymes. Guo said he’s particularly interested “in understanding why enzymes evolved to use a specific metal center to carry out enzymatic reactions and how the geometric coordination environment of the metal center could enable it to perform different types of chemistry.”

Using EPR, which is a method for studying molecules that have unpaired electrons, Guo concluded that the newly discovered halogenase does contain two copper atoms at its center as the UCLA team surmised. The EPR results also suggested that the two copper atoms don't sense each other magnetically.

“At least they don't see each other’s magnetic fields,” Guo said. “So that suggests these two copper sites are not very close to each other.” Guo’s spectroscopic results thus set up the initial entry point to further understand the detailed mechanism of this fascinating enzyme.

With the discovery of the copper-dependent enzyme, the Tang lab expanded enzymatic halogenation reaction that was not possible with the iron-based halogenases. For example, the enzyme is able to perform iodination of an unactivated C-H bond that has not been demonstrated by enzymes. This is due to the compatibility in electronic properties between copper and iodide, which is not feasible with iron. Capitalizing on the unique property of the copper center, other useful functional groups such as thiocyanate and selenocyanate also were shown to be selectively incorporated into the substrate molecule. Computational analysis by Ken Houk’s group at UCLA, including postdoctoral scholar Panpan Chen and graduate student Qingyang Zhou, established a potential mechanism for the copper-dependent halogenation that will be explored in detail in the future.

It turns out this family of copper-dependent enzymes are widely found in nature, most of which catalyze unknown reactions.  The Tang research team is now exploring functions of these previously cryptic enzymes to understand their potentials as biocatalysts.

This research represents a collaborative work from multiple research groups in the Department of Chemistry and Biochemistry, including those of Professors Joseph Loo, Ken Houk and Jose Rodriguez. In addition to CMU’s Guo, Professor Shabnam Hematian from the University of North Carolina at Greensboro also contributed to the work.