C–H Hydroxylation in Paralytic Shellfish Toxin Biosynthesis

Abstract

The remarkable degree of synthetic selectivity found in Nature is exemplified by the biosynthesis of paralytic shellfish toxins such as saxitoxin. The polycyclic core shared by saxitoxin and its relatives is assembled and subsequently elaborated through the installation of hydroxyl groups with exquisite precision that is not possible to replicate with traditional synthetic methods. Here, we report the identification of the enzymes that carry out a subset of C-H functionalizations involved in paralytic shellfish toxin biosynthesis. We have shown that three Rieske oxygenases mediate hydroxylation reactions with perfect site- and stereoselectivity. Specifically, the Rieske oxygenase SxtT is responsible for selective hydroxylation of a tricyclic precursor to the famous natural product saxitoxin, and a second Rieske oxygenase, GxtA, selectively hydroxylates saxitoxin to access the oxidation pattern present in gonyautoxin natural products. Unexpectedly, a third Rieske oxygenase, SxtH, does not hydroxylate tricyclic intermediates, but rather a linear substrate prior to tricycle formation, rewriting the biosynthetic route to paralytic shellfish toxins. Characterization of SxtT, SxtH, and GxtA is the first demonstration of enzymes carrying out C-H hydroxylation reactions in paralytic shellfish toxin biosynthesis. Additionally, the reactions of these oxygenases with a suite of saxitoxin-related molecules are reported, highlighting the substrate promiscuity of these catalysts and the potential for their application in the synthesis of natural and unnatural saxitoxin congeners.

Natural Voltage‐Gated Sodium Channel Ligands: Biosynthesis and Biology

“…Characterization of SxtA supports 12 as a biosynthetic intermediate . However, in vitro reactions with sxt oxygenases suggest that decarbamoyl 11‐α‐hydroxysaxitoxin ( 16 ) is not on the pathway to saxitoxin ( 3 ) and that it is more likely a shunt product of this biosynthetic pathway . The first and final steps in the pathway are well supported thus far, but the critical oxidative cyclization of the linear 12 to form the tricyclic common to the PSTs is yet to be revealed.…”

“…In vitro characterization of the enzymes proposed to be involved in PST biosynthesis, has been explored for five enzymes to date: the polyketide‐like synthase SxtA, Rieske oxygenases SxtT, SxtH, and GxtA, and the sulfotransferase SxtN (Figure A, B) . SxtA performs the first set of reactions in the saxitoxin biosynthetic pathway, assembling intermediate 12 from arginine, S ‐adenosylmethionine, and malonyl‐CoA (Figure A) .…”

“…Fragments of the gene encoding this enzyme have also been identified in dinoflagellates and are often used as a method of detection for PST‐producing capabilities in biological samples . The Rieske oxygenase SxtH was found to install the C12 β‐hydroxyl group prior tricycle formation while Rieske oxygenases SxtT and GxtA perform site‐ and stereoselective C−H hydroxylation reactions at the C12 α‐position of β‐saxitoxinol ( 13 ) to form saxitoxin ( 3 ) and the C11 β‐position of saxitoxin ( 3 ) to form 11‐β‐hydroxysaxitoxin ( 14 ), respectively (Figure B) . The timing of installation of the C12 β‐hydroxy group present in saxitoxin ( 3 ) has not been precisely defined.…”

“…The timing of installation of the C12 β‐hydroxy group present in saxitoxin ( 3 ) has not been precisely defined. However, in vitro experiments have shown that SxtH mediates this hydroxylation prior to tricycle formation . Additionally, the native redox partners necessary for catalysis by the Rieske oxygenases in cyanobacteria have not been identified; the reported study uses a non‐native redox partner, VanB, from the vanillin O ‐demethylase Rieske oxygenase system in Pseudomonas as a surrogate .…”

“…However, in vitro experiments have shown that SxtH mediates this hydroxylation prior to tricycle formation . Additionally, the native redox partners necessary for catalysis by the Rieske oxygenases in cyanobacteria have not been identified; the reported study uses a non‐native redox partner, VanB, from the vanillin O ‐demethylase Rieske oxygenase system in Pseudomonas as a surrogate . Finally, the most recently characterized enzyme, SxtN, was found to catalyze the sulfation of saxitoxin ( 3 ) to form gonyautoxin 5 ( 15 ) .…”