Spying on nature's drug factories
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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