Chemistry Reference
In-Depth Information
6.5 FUNGAL PR-PKS
The domain structure of PR PKSs is much closer to mammalian FAS, with an
N-terminal KS followed by AT, and DH domains (Fig. 6.2d). A so-called “core”
domain follows the DH, which is followed by a KR and the PKS terminates
with an ACP domain. The domain structure differs considerably from the NR
PKS—no SAT domain or PT domain exists, and the PKS terminates after the
ACP and seems not to require a TE/CLC domain. No obvious catalytic machinery
exists for off-loading the product. The DNA sequence of the KS domain is dis-
tinguishable from NR-PKS and FAS KAS domains using selective PCR primers
and DNA probes (3). Overall, however, catalytic domains closely match those
of the mamalian FAS, although the “core” domain is different. Remarkably, the
fungal MSAS genes are closely related to recently discovered
bacterial
genes
for the synthesis of the nonreduced tetraketide orsellinic acid (
1
) (12) apart from
lacking a
∼
450-amino acid region that encompasses the KR domain.
Although many PR PKS genes are known from genome sequencing projects,
only three genes have been matched to chemical products—in all cases, the
tetraketide 6-MSA (
2
) MSAS from
Penicellium patulum
is one of the smallest
Type 1 PKS at 188 KDa. It proved relatively easy to isolate, and many of the
earliest
in vitro
studies of the enzymology of any PKS were carried out with this
enzyme (13, 14). The PKS is evidently programmed—acetate must be extended
three times, and the KR must only act once, after the second extension. In the
absence of NADPH, the KR reaction cannot occur, the tetraketide is not produced,
and triketide lactone (TAL) (
44
) is produced instead (Fig. 6.2d). This reaction
reveals that chain extension and reduction are linked, and it indicates that the
KR must act during chain extension. Mammalian FAS also produces TAL (
44
)
under the same circumstances.
Chain length determination seems to use a “counting mechanism” as in the
case of the Type 2 actinorhodin PKS. Incubation of various acyl CoA starter
units with malonyl CoA with MSAS in the absence of NADPH and acetyl CoA
resulted in two chain extensions to produce the corresponding substituted TALs.
Moriguchi et al. (15) have carried out an interesting series of expression
experiments in the yeast
Saccharoveyces cerivisiae
in which two copies of the
MSAS gene can be expressed simultaneously. This inspection has allowed com-
plementation experiments in which they show that up to 44 amino acids from the
N-terminus can be removed and activity retained. However, at the
C
-terminus,
deletion of as few as nine amino acids caused loss of activity because of removal
of key ACP residues. Use of combinations of deletion mutants, with the knowl-
edge that MSAS forms homotetramers, provided evidence that the ACP of one
peptide chain must interact with the KS of another chain. In addition, a short core
domain region was identified (15). This finding was essential for successful com-
plementation and suggested that it acts as a motif required for subunit-subunit
recognition similarly to the core region of mammalian FAS, which has been
shown to mediate assembly of the synthase.
