Chemistry Reference
In-Depth Information
reactions. As such, an understanding of how KR domains exert stereochemi-
cal control of their hydroxylated product is a critical aspect to deciphering the
mechanism of DH-mediated double bond formation. Through bioinformatic and
biochemical analyses, an appreciation of ketoreductase-influenced stereochem-
istry has emerged (24, 25). Thus, Caffrey (25) has proposed that KR domains
can be divided into two classes, depending on the final configuration of the
β
-hydroxyl moiety. The so-called “A” class generates an L-3-hydroxy prod-
uct, whereas the “B” class produces the D-3-hydroxy polyketide intermediate.
Although little difference exists between these two putative classes at the amino
acid sequence level, the presence of a conserved aspartate residue within an
LDD motif correlates well with “B” class. This motif is absent in the defined
“A” class of KR domains. An additional diagnostic feature of the “A” class of KR
domains is the presence of a conserved tryptophan residue. Recently, Keatinge-
Clay (18) has proposed a refinement of the KR class descriptions as originally
suggested by Caffrey (25), effectively increasing the number of possible KR types
from two to six (18). This new classification takes into consideration whether a
given KR domain (either reductively competent or incompetent) is located in an
epimerization-competent module. Although this new classification offers a more
complete description of PKS KR domains, for simplicity we will continue to use
Caffrey's KR nomenclature throughout our discussion of double-bond formation.
While examples of both D- and L-hydroxyl group configurations can be found
within polyketide natural products, recent evidence suggests that DH domains
require a stereospecific 3-hydroxyacyl intermediate. Bioinformatic analyses per-
formed on 71 KR domains for which the stereochemical outcome of the reduction
is cryptic because of subsequent dehydration revealed that all belong to the “B”
class of KR domains (25). As such, it appears that the generally preferred sub-
strate for DH domains is a D-3-hydroxyacyl chain. However, direct experimental
evidence has been difficult to obtain because the 3-hydroxyacyl intermediate is
transient in modules that contain a DH domain. Recently, biochemical studies of
the DH domain found in module 2 of the pikromycin PKS system (Fig. 7.2b)
(26) have supported this hypothesis; inactivation of the DH domain resulted in
the exclusive generation of the D-3-triketide acylthioester intermediate from a
diketide substrate (27). Aside from this study, no other reports probe the sub-
strate preference or catalytic mechanism of DH domains within PKS systems;
therefore, much of what is known has been elucidated from studies of fatty acid
biosynthesis (28). Previous studies on the dehydration step that is catalyzed by
the yeast fatty acid synthase confirmed the
syn
elimination of water from a D-
(3 R)-hydroxyacylthioester substrate (29). This result is consistent with the stere-
ospecificity of the PKS DH domain and may suggest that
trans
unsaturated bonds,
typically found in polyketides, are likewise formed via
syn
water elimination.
7.2.2 Cis Double Bonds
Although rare, several PKS biosynthetic systems can install
cis
double bonds
into the final polyketide product (Fig. 7.4). Several possible mechanisms could
