Sequencing of the PKS gene cluster in C. violaceus was conducted and confirmed to reveal five type I polyketide synthases and post-PKS tailoring enzymes with O-methyltransferase and cytochrome P450 monooxygenase.
The overall structure correlates well with modular arrangement of PKS-encoded proteins, aside from some unexpected elements that are likely caused by inactive domains.
Initial loading uses a GCN5-related N-acetyltransferase (GNAT) domain instead of the typical AT domain.

Gephyronic acid contains a methyl ether at C-5 and a C-12/C-13 epoxide.
These functional groups are incorporated by post-PKS tailoring enzymes.
GphA is likely responsible for installation of the C-5 methyl ether.
O-methyltransferases SpiB and SpiK used in spirangien biosynthesis exhibit the same SAM-binding motif as GphA.
GphK is a member of the cytochrome p450 superfamily and is suspected to carry out the epoxidation of the C12-C13 olefin.
Such epoxidation in post-PKS modications has been seen in epothilone biosynthesis by EpoK. In EpoK, the consensus mechanism of epoxidation by P450 involves the formation of a pi-complex between an oxoferryl pi-cation radical species (Feᴵⱽ) and the olefin pi bond, followed by electron transfer, formation of the olefin pi-cation radical and finally epoxidation.

However, it is also possible that in addition to cytochrome p450, a FAD-dependent monoxygenase is also required to install the epoxide.
This codependent process is seen in tirandamycin biosynthesis by TamL. Experiments to clarify the function of these enzymes in gephyronic acid biosynthesis are underway.
Sequencing of the PKS gene cluster in C. violaceus was conducted and confirmed to reveal five type I polyketide synthases and post-PKS tailoring enzymes with O-methyltransferase and cytochrome P450 monooxygenase. The overall structure correlates well with modular arrangement of PKS-encoded proteins, aside from some unexpected elements that are likely caused by inactive domains. Initial loading uses a GCN5-related N-acetyltransferase (GNAT) domain instead of the typical AT domain.
Gephyronic acid contains a methyl ether at C-5 and a C-12/C-13 epoxide. These functional groups are incorporated by post-PKS tailoring enzymes. GphA is likely responsible for installation of the C-5 methyl ether. O-methyltransferases SpiB and SpiK used in spirangien biosynthesis exhibit the same SAM-binding motif as GphA.
GphK is a member of the cytochrome p450 superfamily and is suspected to carry out the epoxidation of the C12-C13 olefin. Such epoxidation in post-PKS modications has been seen in epothilone biosynthesis by EpoK. In EpoK, the consensus mechanism of epoxidation by P450 involves the formation of a pi-complex between an oxoferryl pi-cation radical species (Feᴵⱽ) and the olefin pi bond, followed by electron transfer, formation of the olefin pi-cation radical and finally epoxidation.
However, it is also possible that in addition to cytochrome p450, a FAD-dependent monoxygenase is also required to install the epoxide. This codependent process is seen in tirandamycin biosynthesis by TamL. Experiments to clarify the function of these enzymes in gephyronic acid biosynthesis are underway.
Gephyronic acid