Streptozotocin (also known by the trade name Zanosar) is a known antibiotic compound and a drug used to treat metastatic pancreatic islet cell carcinoma. Although a well-known drug, the biosynthetic pathway has remained a black box.

A team of researchers from Harvard University and The Pennsylvania State University have recently untangled the process, publishing the mechanism behind streptozotocin’s production. Led by Emily Balskus, PhD, professor of chemistry and chemical biology, Harvard University, the researchers showed that the compound is produced through an enzymatic pathway and revealed the novel chemistry that drives the process.

The work is described in a February 6 paper titled, “An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin” published in Nature.

What makes the molecule such an effective anticancer agent is a chemical structure known as a nitrosamine—what Balskus called the molecule’s reactive “warhead.” Known to be highly reactive, nitrosamines have been shown to be toxic in a host of other compounds, and are most commonly known outside of cancer treatment as carcinogens found in everything from tobacco to cured meats.

“This chemical motif has a great deal of biological relevance, and has been investigated thoroughly,” Balskus said. “Until our work, the view of how this chemical motif was generated in biological systems involved non-enzymatic chemistry—it was just something that occurred under the right conditions.”

Balskus and colleagues sought to explore whether bacteria evolved a natural pathway to produce nitrosamine compounds. The results were “a big surprise,” noted Balskus, because “it’s made by an enzyme in a very different way than all other known routes to make nitrosamine. The reaction has very limited, if any, precedent in biological or synthetic chemistry.”

What Balskus and colleagues found was that SznF, a metalloenzyme from the biosynthesis of streptozotocin, catalyses an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product. They write that, “structural characterization and mutagenesis of SznF reveal two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors.”

This biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-haem-iron-dependent enzymes to include N–N bond formation. “Both of these domains had been associated with other chemistry in enzymes, but in the context of this protein, both are doing things that are really new,” Balskus said. “So overall, from a purely chemistry perspective, it’s a very exciting enzyme.”

It’s equally exciting from a biological perspective, she adds, because it shows for the first time that biology evolved a specific pathway for manufacturing nitrosamines.

The team found that biosynthetic gene clusters that encode SznF homologues are widely distributed among bacteria—including environmental organisms, plant symbionts, and human pathogens—which suggests an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites. “It seems that we have underappreciated how nature might be using compounds like this,” added Balskus. “The discovery that there are dedicated enzymes for making this type of functional group, and the fact that it may be made by so many types of microbes suggest an important role for its biology.”

Going forward, Balskus will continue to work with collaborators to understand how the enzyme works at the molecular level and to better understand the intermediate steps in the production of nitrosamine. She hopes to investigate whether other bacteria—particularly human pathogens—rely on similar enzymes to produce compounds that are potentially toxic and to further unravel those mechanisms.

“The question we want to answer is whether this new type of enzyme is allowing human pathogens to do something that is damaging the host,” she said. “Now that we’ve found these gene clusters, we can start to ask what these other N-nitrosamine containing compounds might be doing.”

 

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