Chemistry Reference
In-Depth Information
4.3.1 Module or Domain Exchange
When considering the modular buildup of NRPSs, the possibility of altering the
peptide product by insertion, deletion, or exchange of modules seems to be an
obvious approach for the production of new compounds. Because the A domains
determine the specificity of each module, even an exchange of fractions smaller
than whole modules in a synthetase could lead to an altered product. In the past,
various attempts have succeeded using these strategies (24, 25). For instance,
the exchange of an A domain in the surfactin NRPS with other A domains of
both bacterial and fungal origin lead to the formation of the expected variants
of surfactin (26). However, in all of these early studies, the apparent turnover
rates were significantly lower than in the wild-type systems. According to com-
mon understanding of NRPSs, two explanations for the drastically slowed down
synthetic process can be given. First, the borders chosen to dissect and to fuse
the catalytic domains might have been unsuitable. Even though the reoccurring,
highly variable so-called linker regions between each pair of catalytic domains
seem not to exhibit secondary structures, their sequence and length might be crit-
ical for proper inter-domain communication. So far, no structure of any enzyme
consisting of two or more NRPS domains has been published, which makes it
difficult to define the right domain border when preparing a cloning strategy for
fusion or for dissection. Second, the specificity of the C domains might result
in a reduced product turnover. Even though a relaxed specificity for the donor
substrate has been reported, the acceptor site seems to be highly specific, which
discriminates against artificial substrates (27). Both the mode of catalytic action
and the molecular and structural basis for the selectivity are not fully under-
stood for C domains so that a straightforward approach for overcoming these
low turnover rates currently cannot be given.
4.3.2 Changing the Specificity Code for A Domains
Sequence alignments of A domains have revealed that domains activating the
same type of building block share a set of conserved residues in the primary
protein sequence (13). With the A domain's crystal structure, one can find that
these residues form the substrate binding pocket (28). These residues are therefore
referred to as the “selectivity-conferring code” of NRPSs (13). One can now ratio-
nally exchange these sets of residues and can obtain fully functional A domains
with altered substrate recognition. For example, this process has been done for
the first module of the surfactin synthetase srfAA, which activated glutamate
(29). In this case, the only difference in the selectivity-conferring code compared
with a glutamine activating A domain lies in one residue. Thus, the single muta-
tion of Lys239 to Gln239 in the enzyme leads to the desired and predicted shift
of specificity. In another experiment, three residues were altered to change the
substrate recognition of the aspartate activating A domain in srfB2 to asparagine.
The corresponding bacterial strain was shown to produce the expected variant
of surfactin containing asparagine at position 5. Even though this elegant way
