Unusual biosynthetic pathway offers a key to future natural product discovery

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Unusual biosynthetic pathway offers a key to future natural product discovery

Bacteria are master engineers of small biologically useful molecules. A new study by Nature Communications revealed one of the tricks of this microbial trade: synthesizing and later inserting a nitrogen-nitrogen bond, such as a prefabricated part, into a larger molecule.

The discovery was made by a group of chemists from the University of Illinois and Harvard University. Together, they confirmed that two otherwise unrelated compounds, produced by bacteria, shared an unusual set of steps in their biosynthetic pathways. Deciphering this type of biochemical process will facilitate the search for other useful biological compounds.

"It's a molecular or genetic stem if you now want to tackle other new molecules that people have never found before," said Wilfred van der Donk, Richard E. Heckert Chair in Chemistry and Howard Hughes Medical Institute researcher. "We are very excited about what's in the paper and what it allows us to do in the future."

Natural products, substances produced by living beings, have provided us with antibiotics, antifungals, cancer therapies and other important pharmaceutical and industrial compounds; the ongoing exploration of the diverse chemical world of microbes is one of our best hopes for future drug discovery. One of van der Donk's main areas of research is the search for new natural products.

Van der Donk shares this objective with a collaborative research team at the Carl R. Woese Institute of Genomic Biology (IGB), of which he is a member. The Microbial Genomes in Mining Research Team aims to accelerate the hunt for natural products by using the power of next-generation genomic technologies. The tools that bacteria and other microbes use to make natural products are enzymes, specialized proteins encoded by genes. The team's long-term goal is to learn to read bacterial genomes and, from the genes each species has, predict the compounds it is capable of producing.

The team is particularly interested in a class of molecules called phosphonates that has already yielded multiple useful compounds. At the beginning of this study, they wanted to understand which gene products allow a cell to form a key characteristic of a particular phosphonate called fosfazinomycin, a compound with antifungal properties: a chemical bond between two nitrogen atoms. Compounds with reactive nitrogen-nitrogen bonds react readily with other molecules such as DNA and proteins and, as such, may contribute to antimicrobial or anticancer activity.

"We looked for fosfazinomycin as a group for probably a decade because of its very unusual structure, but we didn't know which genes" provided the enzymes to synthesize it, explains van der Donk. "We've decided to agree, let's see how nature creates this nitrogen-nitrogen bond."

After the group began working on the project, two publications by researchers focusing on other natural products described a nitrogen-nitrogen binding process in which one nitrogen atom is integrated into the molecule and another is fixed later - the organism builds the molecule piece by piece, like a child with a package of basic Lego bricks.

Van der Donk's group discovered with surprise that the nitrogen-nitrogen binding of their molecule was not formed in this way. Instead, the bacteria studied created nitrogen nitrogen bonds in a much smaller molecule, such as a specialized Lego part, and later installed this part in the larger molecule that would become fosfazinomycin.

"As we continued to work, we realized that things in our system are very different," said Mr. van der Donk. "In our case, it seemed as if nature was making this molecule containing a nitrogen-nitrogen bond as a prepackaged molecular entity that was then released into an existing biosynthetic pathway."

The research project took another fortuitous turn when Kwo-Kwang (Abraham) Wang, a graduate student and first co-author, presented the preliminary results at a conference. He was approached by Tai Ng, a Harvard chemistry graduate student, who, with his laboratory group led by Professor Emily Balskus, was studying a natural product and a promising anti-cancer agent called kinamycin. Kinamycin contains a nitrogen-nitrogen bond, and Ng's research suggests that it also shares the suspected prefabrication step for fosfazinomycin.

"We had noticed that their molecule[is synthesized using] the same genes, but we didn't really know how it integrated either, because they make a structure containing a completely different nitrogen-nitrogen bond that doesn't look anything like our molecule," says van der Donk. The two groups began working together, coordinating experiments in which labelled molecules were given to bacteria capable of synthesizing each of the two natural products, to see which intermediate molecular structures could be transparently introduced into the natural biosynthesis pathway in the cell.

"We made these labelled compounds, gave them to the producing organism, isolated the final product, for Harvard's kinamycin group and for us, fosfazinomycin, and saw if the nitrogen nitrogen portion of the molecules we gave to these organisms was installed in the final product," said van der Donk. "We did it for four different compounds and each time the answer was yes, yes, yes, yes, yes, yes, yes."

The discovery of this improbable commonality in the way two dissimilar molecules are produced has increased researchers' confidence in the functional roles of the genes involved. They now have a new genomic signature to add to their lexicon, something they can look for in other bacterial genomes while continuing the search for useful natural products.

"We need to learn more about how natural products are made. This is an excellent example; now that we know, we can use this knowledge. Before that, it was just a bunch of genes and we didn't really know what to do with them," says van der Donk. "By attacking unknown gene groups[we hope to be able to] see immediately from the gene group, it must be a new molecule. Could this molecule be the next antibiotic or the next antitumor?"