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. 2018 Sep 19;140(37):11863-11869.
doi: 10.1021/jacs.8b08901. Epub 2018 Sep 7.

C-H Hydroxylation in Paralytic Shellfish Toxin Biosynthesis

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C-H Hydroxylation in Paralytic Shellfish Toxin Biosynthesis

April L Lukowski et al. J Am Chem Soc. .
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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.

Figures

Figure 1.
Figure 1.
(A) Natural products derived from saxitoxin (STX, 9). The molecules with the highest and lowest Kd are outlined. *The Kd of 11-β-hydroxysaxitoxin (7) has not been determined, but its IC50 value ranks the molecule below saxitoxin in terms of affinity. (B) Previously proposed STX (9) biosynthetic pathway. (C) Saxitoxin gene cluster and potential enzymes involved in late-stage oxygenation.
Figure 2.
Figure 2.
Methods for Fe(III)-hydroperoxo generation in Rieske oxygenases. (A) Electrons supplied by a protein redox partner to the Rieske iron-sulfur cluster and subsequent reduction of O2 at the mononuclear iron binding site. (B) Direct generation of Fe(III)-hydroperoxo species from hydrogen peroxide.
Figure 3.
Figure 3.
Reactions of SxtT and GxtA. (A) Productive hydroxylation of ddSTX (12) with SxtT in the presence of H2O2. (B) Efficiency of the SxtT hydroxylation of ddSTX (12) with non-native redox partners. (C) Reactivity of SxtT homologs. (D) GxtA hydroxylation of STX (9).
Figure 4.
Figure 4.
(A) Reactions of SxtT with various substrates: hydroxylation of dc-ddSTX (11) to dc-α-STOH (15), hydroxylation of dc-β-STOH (16) to dc-STX (1), hydroxylation of ddSTX (12) to α-STOH (14), and hydroxylation of β-STOH (13) to STX (9). (B) Reactions of GxtA with various substrates: hydroxylation of dc-ddSTX (11) to decarbamoyl 11-β-saxitoxinol (17), hydroxylation of dc-α-STOH (15) to decarbamoyl 11-β-hydroxy-α-saxitoxinol (18), hydroxylation of dc-β-STOH (16) to dc-11-β-hydroxy-β-saxitoxinol (19), hydroxylation of dc-STX (1) to dc-11-β-hydroxysaxitoxin (20), hydroxylation of ddSTX (12) to 11-β-saxitoxinol (21), hydroxylation of α-STOH (14) to 11-β-hydroxy-α-saxitoxinol (22), hydroxylation of β-STOH (13) to 11-β-hydroxy-β-saxitoxinol (23), hydroxylation of STX (9) to 11-β-hydroxysaxitoxin (7), and neoSTX (4) to 11-β-hydroxy neosaxitoxin (24). TTNs were determined using reactions consisting of 5 μM SxtT or GxtA, 5 μM VanB, 200 μM substrate, 500 μM NADH, 100 μM Fe(NH4)2(SO4)2, and 50 mM TrisHCl pH 7.0 incubated at 30 °C for 2 h.
Figure 5.
Figure 5.
(A) Reaction of SxtH with arginine methyl ester (25) to generate hydroxylated arginine methyl ester (26) and LC/MS trace compared to standard. (B) MS/MS comparison of 26 standard and product of the SxtH reaction. (C) Scheme mapping a hypothetical SxtH product (27) onto STX (9), highlighting the position of hydroxylation in the final product.
Scheme 1
Scheme 1
Revised biosynthetic hypothesis.

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