Stereospecific Formation of E- and Z-Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase

Abstract

The dehydratase domain FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzed the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP1 (5) and (E)-2-butenoyl-FosACP1 (11), as established by a combination of direct LC-MS/MS and chiral GC-MS. FosDH1 did not act on either (3S)-3-hydroxybutyryl-FosACP1 (6) or (Z)-2-butenoyl-FosACP1 (12). FosKR2, the ketoreductase from module 2 of the fostriecin PKS that normally provides the natural substrate for FosDH2, was shown to catalyze the NADPH-dependent stereospecific reduction of 3-ketobutyryl-FosACP2 (23) to (3S)-3-hydroxybutyryl-FosACP2 (8). Consistent with this finding, FosDH2 catalyzed the interconversion of the corresponding triketide substrates (3R,4E)-3-hydroxy-4-hexenoyl-FosACP2 (18) and (2Z,4E)-2,4-hexadienoyl-FosACP2 (21). FosDH2 also catalyzed the stereospecific hydration of (Z)-2-butenoyl-FosACP2 (14) to (3S)-3-hydroxybutyryl-FosACP2 (8). Although incubation of FosDH2 with (3S)-3-hydroxybutyryl-FosACP2 (8) did not result in detectable accumulation of (Z)-2-butenoyl-FosACP2 (14), FosDH2 catalyzed the slow exchange of the 3-hydroxy group of 8 with [¹⁸O]-water. FosDH2 unexpectedly could also support the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP2 (7) and (E)-2-butenoyl-FosACP2 (13).

Figure 1

Fostriecin (1) and related polyketides.

Figure 2

Biosynthesis of fostriecin (1) by a modular polyketide synthase. PKS domains: ketosynthase (KS), acyl transferase (AT), acyl carrier protein (ACP), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), thioesterase (TE).

Figure 3. Hydration of (2Z,4E)-3-hydroxy-4-hexenoyl-FosACP2 (21) by FosDH2

Chiral GC-MS of methyl (3R,4E)-3-hydroxy-4-hexenoate from incubation of 21 with FosDH2. A-1: XIC m/z 71); A-2: Co-injection with added methyl (3S,4E)-3-hydroxy-4-hexenoate, XIC m/z 71. See Figure S19 for comparisons of the TIC of reaction product, rt 36.89 min, and authentic methyl (3R,4E)-3-hydroxy-4-hexenoate.

Scheme 1

Synthesis of C4 acyl-FosACP substrates.

Scheme 2

Synthesis of C6 acyl-FosACP2 derivatives.

Scheme 3

Stereospecific dehydration and hydration by FosDH1.

Scheme 4

Stereochemistry of FosKR2-catalyzed reduction.

Scheme 5

FosDH2-catalyzed hydration and dehydration of FosACP2-bound triketide substrates.

Scheme 6

FosDH2-catalyzed hydration and dehydration of FosACP2-bound diketide substrate analogs.