Chemistry Reference
In-Depth Information
6.4.1 NR-PKS Loading Component
Feeding experiments with isotopically labeled precursors have shown that many
NR fungal polyketides are formed by the use of “advanced” starter units. In the
classic case of norsolorinic acid (
7
) biosynthesis, it has long been known that
hexanoate forms the starter unit. Differential specific incorporation of acetate into
the early and late positions in compounds such as citrinin (
3
) have been used to
argue that these compounds may have been formed by more than one PKS so
that one PKS makes an advanced starter unit, which is passed to a second PKS
for additional extension.
Evidence for this suggestion is growing. Townsend has defined the molecular
basis for the ability of NR-PKS to use starter units derived from other FAS or
PKS systems. Two genes in the aflatoxin biosynthetic gene cluster of
Aspergillus
parasiticus
(
stcJ
and
stcK
) encode the
α
and
β
components of a typical fungal
FAS (HexA and HexB). Clustering of these FAS genes with the NSAS PKS
suggested that HexA and HexB probably produced hexanoate for use as the
norsolorinic acid starter unit. The protein complex formed between NSAS, HexA,
and HexB, which is known as NorS, was isolated and characterized (6). This
1.4-MDa protein complex synthesizes norsolorinic acid (
7
) from malonyl CoA,
acetyl CoA, and NADPH. Townsend showed that hexanoyl CoA is not a free
intermediate produced by NorS, which suggests that hexanoate produced as an
ACP derivative by the HexA/HexB FAS must be passed directly to NSAS. In
the absence of NADPH, hexanoate cannot be formed by the FAS components;
thus, no norsolorinic acid is formed.
The N-terminal domain of NSAS posesses canonical acyl transferase sequence
motifs so that this domain could be a candidate for the required starter unit
transferase. This domain was cloned and expressed along with the ACP and was
shown to catalyze the transfer of hexanoate from CoA onto the the ACP. Site-
directed mutagenesis experiments to remove the proposed catalytic cysteine of
the transferase resulted in loss of catalytic activity. The N-terminal transferase
showed significant selectivity for the transfer of hexanoate over longer or shorter
acyl chains (7). Thus, the N-terminal domain of NSAS acts as a starter unit:ACP
transacylase (SAT) component.
Sequence comparison with other known NR PKS suggests that such SAT
domains are common. In the few cases where the PKS sequence has been cor-
related with product structure the presence of SAT domains now explains prior
results from feeding experiments, which suggested the use of advanced starter
units. For example it is now known (8) that two PKS genes are involved in
the biosynthesis of zearalenone (
12
)—one of these is a HR-PKS and probably
provides a highly reduced hexaketide as a starter unit. The second zearalenone
PKS is a NR-PKS possessing an N-terminal SAT domain, which likely loads the
hexaketide ready for three further extensions. Most NR PKS seem to possess
potential SAT domains whether they require an acetate starter unit or not. For
example, polyketide synthases involved in the biosynthesis of YWA1 (
39
)(WAS)
and tetrahydroxynaphthalene (THNS). In the case of THNS from
C. lagenarium,
