Vinylogous Dehydration by a Polyketide Dehydratase Domain in Curacin Biosynthesis

Abstract

Polyketide synthase (PKS) enzymes continue to hold great promise as synthetic biology platforms for the production of novel therapeutic agents, biofuels and commodity chemicals. Dehydratase (DH) catalytic domains play an important role during polyketide biosynthesis through the dehydration of the nascent polyketide intermediate to provide olefins. Our understanding of the detailed mechanistic and structural underpinning of DH domains that control substrate specificity and selectivity remains limited, thus hindering our efforts to rationally re-engineer PKSs. The curacin pathway houses a rare plurality of possible double bond permutations containing conjugated olefins as well as both cis- and trans-olefins, providing an unrivaled model system for polyketide dehydration. All four DH domains implicated in curacin biosynthesis were characterized in vitro using synthetic substrates and activity was measured by LC-MS/MS analysis. These studies resulted in complete kinetic characterization of the all trans trienoate-forming CurK dehydratase, whose kcat of 72 s−1 is more than three-orders of magnitude greater than any previously reported PKS DH domain. A novel stereospecific mechanism for diene formation involving a vinylogous enolate intermediate is proposed for the CurJ and CurH dehydratases based on incubation studies with truncated substrates. A synthetic substrate was co-crystallized with a catalytically inactive Phe substitution in the His-Asp catalytic dyad of CurJ DH to elucidate substrate-enzyme interactions. The resulting complex suggested the structural basis for dienoate formation and provided the first glimpse into the enzyme-substrate interactions essential for the formation of olefins in polyketide natural products. This examination of both canonical and non-canonical dehydration mechanisms reveals hidden catalytic activity inherent in some DH domains that may be leveraged for future applications in synthetic biology.

Structural and Functional Trends in Dehydrating Bimodules from trans-Acyltransferase Polyketide Synthases

“…The thioester could shift towards the surface of the protein to form a hydrogen bond with a histidine conserved in DHs from type B dehydrating bimodules (termed H” here, in the Hxx H GxxxxP motif); this would facilitate the abstraction of a proton from the l -γ-hydrogen by the same histidine that abstracted the proton from the l -α-hydrogen ( H xxHGxxxxP) while the aspartate helps donate a proton to the l -δ-hydroxy leaving group. This vinolygous dehydration could be similar to that proposed for two DHs from the curacin cis -AT PKS (Route 1 in Figure 3b) (Fiers et al, 2016). However, a conformational change would be necessary since the side chain of H” is observed on the surface of the DH domain.…”

“…If the DH of type B dehydrating bimodules is mechanistically equivalent to canonical DHs, it would be expected to only dehydrate β-hydroxyacyl intermediates and thus operate first on the KR A -generated l -β-hydroxyacyl intermediate bound to ACP of the first module and then on the KR B -generated d -β-hydroxyacyl intermediate bound to ACP of the second module (Keatinge-Clay, 2016). However, recent analysis of DHs from the curacin cis -AT PKS have revealed that some DHs can perform the double dehydration of a d -β- l -δ-dihydroxyacyl-ACP substrate, possibly through a canonical dehydration to yield an α/β- trans unsaturated intermediate and a subsequent vinolygous dehydration (Fiers et al, 2016). The DHs of type B dehydrating bimodules may perform double dehydration similarly.…”

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

“…18 The structure of the E. coli dehydratase (FabA) displays a characteristic hotdog fold that has been observed in all other DH structures from both FAS and PKS systems. 7a,19,20 The active site of each DH harbors a universally conserved pair of His and Asp residues. Schwab, in a critical review of research on FabA and the closely related dehydratase-isomerase FabZ, has discussed a one-base, two-step mechanistic model in which the active site imidazole residue first catalyzes the stereospecific removal of 2-H si of the 3-hydroxyacyl-ACP substrate following which the transiently-generated imidazolium species donates its proton to the 3-hydroxyl group to promote C–O bond cleavage.…”

“…9d,20 While one or two of the four DH domains from the curacin PKS are thought to be responsible for formation of unsaturated intermediates possessing ( Z ) double bonds, all four proteins exhibit the common double hotdog fold and high levels of mutual structural homology, while the actual enzymatic formation of ( Z )-enoyl-ACP products has not yet been reported. 7 FosDH2 shows 71.0% mutual sequence identity (88.8% similarity) over 276 aa to the closely related PlmDH1 from module 1 of the phoslactomycin PKS, which has also been implicated in the formation of a cis double bond (Figure S32). Interestingly, FosDH1 and FosDH2 themselves show a more modest 40.8% mutual sequence identity (63.4% similarity), while retaining each of the key conserved amino acid motifs typified by the structurally characterized dehydratases EryDH4 and RifDH10.…”

“…14a There are already sufficient exceptions 7b,8b,c,9d,23,25 to this superficially appealing generalization to establish that it does not have reliable predictive value.…”