Metamorphic enzyme assembly in polyketide diversification

Abstract

The chemical diversity of natural products is fueled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism [1][2][3][4][5] . However, co-evolution of enzymes as functional assemblies for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel enzyme assemblies with an extraordinarily high sequence identity form a β-branched cyclopropane in the curacin A (Cur), and a vinyl chloride group in the jamaicamide (Jam) pathways, respectively. The assemblies include a halogenase (Hal), a 3-hydroxy-3-methylglutaryl (HMG) enzyme cassette for β-branching and an enoyl reductase domain (ER). Bioinformatic analysis indicated that the corresponding genes were inserted into modular polyketide synthases (PKSs) via acyltransferase (AT) domain replacement. The Hal from CurA, and the dehydratases (ECH 1 s) and decarboxylases (ECH 2 s) within the HMG enzyme cassettes and ERs from both Cur and Jam were assessed biochemically to determine the mechanism of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide β-branching pathway was modified by introduction of a γ-chlorination step on (S)-HMG mediated by Cur Hal, a nonheme Fe II , α-ketoglutarate (α-KG)-dependent halogenase 6 . In a divergent scheme, Cur ECH 2 was found to catalyze formation of the α,β C=C enoyl thioester, whereas Jam ECH 2 formed a vinyl chloride moiety by selectively generating the corresponding β,γ C=C (enoyl thioester) of the 3-methyl-4-chloroglutaconyl decarboxylation product. A non-conserved Tyr 82 residue in Cur ECH 2

Conformational switch triggered by α-ketoglutarate in a halogenase of curacin A biosynthesis

“…4A). Hal is highly specific for its substrate, requiring both the S-hydroxyl group at C3 and the C5-carboxylate (8). In the model, the S-hydroxyl group is hydrogen bonded to the Ser120 side chain, which forms a hydrogen bond with Cl − in structures containing αKG ( Fig.…”

“…These residues include three basic and two polar residues that may recognize (S)-HMG-ACP (Leu112, Lys48, Lys50, Lys54, Ser44, and Tyr68), and four active-site residues that directly or indirectly contact αKG or chloride (Ser120, Ser132, Arg241, and Arg247). Production of 4-Cl-HMG-ACP from (S)-HMG-ACP was monitored by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), including a phosphopantetheine (PPant)-ejection assay (19), which releases Cl-HMG-PPant from Cl-HMG-ACP (8). Catalytic efficiency was monitored by the relative abundance of Cl-HMG-PPant and HMG-PPant, which differ in m∕z by 34 units (Fig.…”

“…1A) (8). The chlorinated product reacted sequentially with the CurE ECH 1 dehydratase, the CurF ECH 2 decarboxylase, and the CurF enoylreductase (ER).…”

“…Following ECH 1 ∕ECH 2 -coupled dehydration and decarboxylation of 4-Cl-(S)-HMG-ACP, CurF ER catalyzed formation of the cyclopropane ring by nucleophilic displacement of chloride (8) on the ACP-bound substrate. As opposed to cyclopropane ring formation, the homologous JamE Hal, JamI ECH 1 , and JamJ ECH 2 enzymes generated a vinyl chloride functional group as the endpoint of the reaction series (8).…”

Asymmetric Reductive Carbocyclization Using Engineered Ene Reductases

“…According to the accepted mechanism, a hydride is delivered from the reduced flavin mononucleotide (FMN) cofactor to the β‐carbon to form an enolate, which is subsequently protonated with the assistance of Tyr‐OH as a proton source 9. We envisioned the use (or modification) of this enzyme family for reductive carbocyclizations by offering substrates that exhibit an additional internal electrophile and thus react intramolecularly with the generated enolate intermediate to produce cyclized products 10, 11. Toward this goal, carbonyl groups, alkylhalides, or epoxides could be used as the electrophile (Scheme 1).…”