DISCUSSION
Assembly line PKSs, which represent some of the largest known proteins,
contain modules and domains connected by loops that are experimentally
challenging to study. To better understand the dynamics of PKSs and
facilitate their engineering, we sought to better characterize these
loops. As the insertion of repetitive sequences naturally occurs
throughout the DNA encoding PKS assembly lines, cataloguing where
insertions persist provides a measure for the tolerance of the encoded
loops to modification.
Out of all of the loops in assembly line PKSs, the most tolerant to
insertion is at the updated module boundary, between KS and
FSD[4-6]. Even 137 unstructured residues at this location seems not
to impair the function of the nannocystin module NcyB_1c. Connecting
modules at this junction may preserve how its component domains
structurally and functionally work together, as supported by recentin vivo and in vitro PKS engineering studies [29, 30].
Although this junction is upstream of the AT domain, AT swapping has
been naturally observed and accomplished through engineering (by
including FSD)[4, 31]. More studies are needed to elucidate the
connection between the AT region and the downstream domains of the
module.
The AT-DH loop of γ/δ-modules is more sensitive to modification than the
AT-KR loop of β-modules. Out of 599 examined modules, there are no
insertions in the AT-DH loop, nor are there deviations from its length
of 5-7 residues when n1 is present. One explanation is that this loop is
structured at the interface between KS and DH. This would mean that this
connection is not hinge-like as in the highly related metazoan fatty
acid synthase[32]. Other connections between enzymatic domains, in
particular DH-KRs and ER-KRc, are also
resistant to modification. Perhaps these loops help position the
catalytic sites of the module relative to ACP.
The loops upstream and downstream of ACP (15 ± 5 and 13 ± 6 residues,
for β-δ modules) may need to be short to prevent docking with
non-cognate enzymes, such as AT of the downstream module; however, these
tethers limit the degrees of freedom available to ACP. Surprisingly,
δ-modules, which contain the most enzymatic domains, possess the
shortest linkers (8 ± 1 and 14 ± 2 residues). As ACP cannot stretch to
access its AT in the assembly line as presented (Figure 1), AT may need
to access another conformation. One possibility is that ATs adopt the
position observed in the electron microscopy reconstructions of a
construct from the pikromycin PKS[8]. Another possibility is that a
hinge between AT and the processing enzymes (immediately after the
LPTYxFx5W motif) facilitates these docking
interactions[32]; however, this requires large, asymmetric motions
throughout the assembly line that would hamper polyketide production
compared to a more rigid, symmetric assembly line. In the yeast fatty
acid synthase, active sites are fixed around reaction chambers in which
the only mobile domain is ACP[33].
Those studying and engineering a PKS may wish to make changes to its
polypeptides. Sequence alignments do not always sufficiently indicate
the tolerance of loops to modification. As the repetitive sequences
catalogued here are not likely to be functionally relevant, they serve
as beacons illuminating where changes can be made both between and
within domains. The reported repetitive sequence insertions could help
in positioning 3C protease cut sites on each end of an ACP to liberate
it from an assembly line for analysis by mass spectrometry, installing
purification tags upstream of Class 1a NDDs or
elsewhere, and adding domains such as fluorescent proteins or polyketide
processing enzymes at desired locations.
As essential as loops are to the proper functioning of assembly line
PKSs, they have been relatively ignored compared to PKS domains.
However, they are informative as to how far domains can move from one
another and provide restraints for structural biologists attempting to
elucidate assembly line architecture. Some loops may be more structured
than previously thought, such as the residues at the AT/DH interface and
those downstream of KRs from γ/δ-modules. With the abundant sequence
information that is now available the tolerance of loops to modification
can be evaluated through bioinformatics. The resistance of domains such
as ACP and KS to these modifications also reveals the importance of
their surfaces for domain-domain and docking interactions. Complementing
what is known about PKS domains by studying the loops that connect them
and are present within them has advanced our understanding of PKS
assembly lines as well as our ability to engineer them.