Naturally occurring complex molecules make attractive research targets, especially if they have some compelling property like antibiotic behaviour. University of British Columbia chemistry professor Katherine Ryan has spent much of her career examining such compounds, which has stoked her curiosity about a key aspect of how these intricate organic structures form.

“It has been this interesting conundrum,” Ryan says, pointing to the nitrogen-nitrogen bond that characterizes more than 200 natural product molecules. “How can nature reverse the reactivity of one of the nitrogens to form a nitrogen-nitrogen bond?”

Besides making up 78 percent of the air we breathe, nitrogen atoms can form stable triple bonds with one another, making N2 one of the most stable of molecules. An established biological route to generate nitrogen gas is via anammox bacteria, which use enzymes employing iron-based cofactors to combine nitrates and ammonia, yielding nitrogen gas and water. But whether similar pathways could yield nitrogen-nitrogen (N-N) bonds in natural products like antibiotics has been unknown. 

Ryan’s group was especially interested in the formation of a unique amino acid called piperazate. It is not found in proteins but instead in a variety of naturally produced molecules called non-ribosomal peptides, many of which have antibiotic properties. Ryan and her colleagues Yi-Ling Du, Hai-Yan He and Melanie Higgins have shed new light on the question of how the N-N bond is formed in piperazate, reporting their findings in Nature Chemical Biology, where they describe the discovery of an enzyme that drives formation of the single N-N bond found in piperazate. 

“People have been doing a lot of genome sequencing of bacteria that make different natural products,” says Ryan, who began exploring gene clusters that were linked with piperazate. One particular gene had long been regarded as encoding a transcriptional regulator. After Du purified and examined this enzyme, however, it turned out to be an enzyme responsible for creating piperazate’s N-N bond. 

Ryan points to the enzyme’s use of an iron-based cofactor to sustain a reaction, similar to what was observed from anammox bacteria. “One exciting implication of this finding is that the chemistry involved in making the N-N bond in piperazate mimics what is thought to occur in anammox bacteria,” she says, suggesting that this may be another example of a natural strategy that employs an iron-based cofactor to form the nitrogen-nitrogen bond.