Chemistry Reference
In-Depth Information
Genetic characterization of the nonactin gene cluster has revealed that an ACP-
less Type II PKS is used to assemble the macrotetrolides (21). The non-PKS is
highly unusual, catalyzing both C-C and C-O bond-forming reactions, function-
ing noniteratively and acting on acyl CoA substrates (which is typical of Type III
PKS). Twenty-three non genes were identified, which includes five discrete KS
proteins, NonJKPQU, and four discrete KR proteins, NonEMNO. Heterologous
expression of these 23 nongenes in
S. lividans
resulted in macrotetrolide pro-
duction, thereby confirming that only these genes are required for macrotetrolide
biosynthesis; sequestration of an ACP from elsewhere on the
S. griseus
genome
does not occur.
The proposed biosynthetic pathway (Fig. 8.3g) requires three condensation
and four reduction steps that would require at least three KS and four KR
genes. It has not been established whether the five KS and four KR proteins
function independently, but it has been demonstrated that all nine enzymes are
required for macrotetrolide biosynthesis. Shen and Kwon (21) have suggested that
the macrotetrolide PKS comprises noniteratively functioning subunits, which is
unprecedented in all Type II PKSs known to date. NonJ and NonK have been
assigned roles in dimerization (see below), which leaves NonPQU to carry out
the three C-C bond-forming reactions. A mutant lacking the NonPQU genes was
able to convert nonactic acid into nonactin, whereas deletion of NonJK resulted
in no macrotetrolide formation. Hence, NonPQU represents a novel Type II min-
imal PKS that acts noniteratively, does not have an ACP, and uses acyl CoA
substrates directly for polyketide biosynthesis.
NonJ has been shown to catalyze the first dimerization step to give (
−
)-
non-actyl-(
)-non-actyl CoA
44
, and NonK is responsible for the stereo-specific
cyclodimerization to afford nonactin (21). All C-C bond forming KSs are
characterized by a Cys-His-His (Asn) catalytic triad (including NonPQU),
whereas NonKJ are characterized by a mutated catalytic triad: Cys-Gly/Tyr-His.
Replacement of the conserved cysteine residue in NonJ or NonK with glycine
gave mutants that could not catalyze the transformation of (
+
)-nonactic acid
into nonactin. Taken together, NonJK cannot function as a decarboxylase and
instead catalyze the C-O bond-forming steps in nonactin biosynthesis using
the same active site cysteine that other KSs use for C-C bond formation. In
common with NonPQU, NonJK act noniteratively on CoA substrates.
The product of the minimal PKS,
41
, is the branch point of the pathway
that diverges into a pair of enantio-specific pathways, each of which involves
two KRs and affords (
+
)- or (
−
)-
42
. The nonactate synthase, NonS, catalyzes
the intramolecular Michael addition of (
±
)-
42a
.
A nonS mutant supplemented with (
±
)-nonactic acid could produce nonactin,
monactin, and dinactin, but not trinactin and tetranactin, which require one/two
(
−
)-homononactate moieties, respectively. This process suggests that NonS can
cyclize (
−
)-nonactic acid
43a
from (
−
)-homononactate
35
only and that
another nonactate synthase is required for the cyclization of (
−
)-
42a
into (
−
)-nonactate
43a
and (
−
+
)-
42b
to give
(
+
)-nonactate
43b
and (
+
)-homononactate.
