Researchers have re-engineered a bacterial enzyme to transform abundant alkanes into industrially important nitrogen-containing compounds that can be used to synthesize pharmaceuticals. In principle, the enzyme, now called P411CHA, could help streamline some methods for creating synthetic molecules, which often rely on multiple catalysts that contain expensive, inefficient precious metals.
“The big picture here is that native enzymes can be repurposed to do chemistry that only humans thought they could do,” says study author Frances Arnold, professor of chemical engineering, bioengineering, and biochemistry at the California Institute of Technology. “We want to expand the universe of chemical reactions that enzymes can catalyze.”
In the recent Nature Chemistry study, the researchers sought an enzyme that would catalyze the insertion of nitrogen atoms into the normally unreactive carbon-hydrogen bonds of molecules more efficiently and sustainably than other catalysts. The process, called amination, creates benzylamines—organic molecules that are common precursors in the synthesis of medicines.
The researchers started with a bacterial enzyme called cytochrome P450BM3, an iron-containing catalyst that normally inserts oxygen into C-H bonds, which had been exposed to multiple rounds of mutagenesis. Screening for enzymatic activity revealed that a version of cytochrome P450BM3, which had accumulated at least 17 different mutations, had gained the ability to aminate the normally inert C-H bonds of alkane molecules.
Additional rounds of directed mutagenesis improved P411CHA’s turnover and its yield of benzylamines to as high as 86 percent for a variety of alkanes. And the enzyme produced most of the benzylamine molecules in one molecular configuration—a feature known as enantiomeric purity—which is critical in order for the benzylamine to have a specific function in biology, for instance as a drug.
“People have struggled to make enantioselective C-H bond aminations,” says John Hartwig, professor of chemistry at the University of California, Berkeley, whose lab investigates new methods for catalyzing synthetic reactions. P411CHA surpasses the turnover and enantioselectivity of many small molecule catalysts, he notes.
The P411CHA enzyme catalyst has the potential to improve upon such catalysts in several other ways. Although non-enzymatic small molecule catalysts are capable of performing C-H bond aminations, most contain expensive precious metals, produce low enantiomeric purity, and display a low turnover. “We want to have clean, sustainable processes that don’t use precious metals and that don’t produce toxic waste,” says Arnold. “If you want clean chemistry, you want to use earth abundant metals.”
A P411CHA-mediated reaction can also take place at room temperature in water in E.coli whole cells or in vitro. And whereas some other catalysts rely on the presence of nitrogen in the molecule that’s being chemically transformed, the new method instead gathers and introduces nitrogen atoms from an external source into the C-H bond—this means that P411CHA can synthesize important nitrogen-containing compounds from simpler nitrogen-free molecules such as alkanes, which contain an abundance of C-H bonds.
The study did reveal certain limitations. P411CHA only works on certain types of alkanes below a certain size threshold and displays differing functionality depending on where the C-H bond is located in the molecule. “This initial paper shows that there’s potential to do enzymatic amination. Biology doesn’t do this normally,” says Hartwig. “It shows promise,” he adds, noting that it’s always a long way from such initial findings to a manufacturing process.