2 October 2002 18:00 EST

by Martina Habeck

Bacteria and fungi are a rich source of natural products that are of therapeutic interest. Many of these are used in the clinic, including penicillin, vancomycin, cyclosporin and bleomycin. The building blocks of these compounds are amino acids and carboxylic acids that are assembled by giant proteins called non-ribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs). These proteins have a modular structure, and each module acts as an independent multifunctional enzyme that joins one amino acid/ carboxylic acid to the growing polypeptide/polyketide chain and makes modifications possible. The specific order of the modules defines the sequence of the incorporated building blocks.

Many scientists have dreamed of redesigning this assembly line in order to create new products with therapeutic activity. The idea is to recombine the modules and thus arrive at a novel compound - a process known as combinatorial biosynthesis.

"In principle, you could do this combination at the DNA level," said Christopher Walsh of Harvard Medical School. He pointed out that scientists can already tell from looking at the DNA sequence whether something will be a PKS or an NRPS assembly line, and even which particular acyl or aminoacyl monomer each module will select.

However, in order to engineer new PKS and NRPS proteins successfully, one also needs to understand the three-dimensional structure of these drug factories. It is well established that PKSs are only active as homodimers. The same holds true for fatty acid synthases, closely related megasynthases that use an assembly-line strategy equivalent to that of the PKSs. Therefore, people assumed that NRPSs would also be oligomeric - but a new study published in Chemistry & Biology says otherwise.

Teams led by Walsh and by Mohamed Marahiel at the University of Marburg in Germany looked at modules from the gramicidin S, tyrocidine and enterobactin biosynthetic systems and investigated their architecture with biophysical and biochemical techniques, such as cross-linking, gel filtration, analytical ultracentrifugation and mutant complementation experiments. No matter what method they used, the answer was the same: "We always find they are monomers," said Walsh.

Ben Shen at the University of Wisconsin-Madison was impressed with the thoroughness of their approach: "Many of the conclusions people draw in the field [are based on] only one set of experiments, and sometimes [the results] are over-interpreted. But this group of scientists used both biochemical and biophysical methods, and even within each category, they have done every single experiment you can think of, and they come to the same conclusion. I think their result is very sound." Since they studied three different NRPS systems, Shen believes their finding is generally applicable.

Where does that finding leave scientists who wish to engineer new "natural" products? According to Shen, PKS engineering gained much momentum with the discovery of the dimeric structure of these proteins. He predicts that Walsh's and Marahiel's discovery "should have the same effect and help us engineer peptide synthetases for new structures."

The finding is important for another reason: Some natural products of therapeutic interest are polyketide and polypeptide hybrids. Among those hybrids are the anticancer drugs bleomycin and epothilone; the latter is currently in clinical trials. This suggests that the polyketide and NRPS assembly lines have to be able to mix and match. But how exactly does that work? How does a dimer interact with a monomer at the molecular level?

That is what the Walsh and Marahiel teams are now investigating, for example by studying the epothilone biosynthetic pathway in more detail. The epothilone assembly line consists of a PKS subunit (epoA), followed by an NRPS subunit (epoB), followed by another PKS subunit (epoC). In other words, there are two PKS/NRPS switch points. "We are trying to understand how A and B interact and how B and C interact," said Walsh. "If we understand the rules of compatibility between the module boundaries, maybe we could swap in different modules and make new versions of the natural product."

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