A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

Abstract

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

Figure 1. The stambomycin structures, their biosynthetic gene cluster in S. ambofaciens ATCC23877 and the modular PKS it encodes.

(a) Structures of stambomycin A-D. The major constituents of the complex, stambomycins A-D (1-4), differ in the nature of their C-26 side chains. (b) Organization of the stambomycin biosynthetic gene cluster, highlighting the location of the samR0483 gene encoding an acyl-CoA carboxylase β-subunit proposed to be involved in unusual extender unit biosynthesis. (c) Module and domain organization of the stambomycin PKS and structure of the polyketide chain it assembles. Carbon atoms derived from malonyl-CoA extender units are highlighted in red, while those derived from a methylmalonyl-CoA starter unit and methylmalonyl-CoA extender units are highlighted in purple. The carbon atoms derived from the unusual extender units utilized by PKS module 12 (boxed) are highlighted in blue (R¹, R² and R³=Me or H, depending on which of the unusual extender units is incorporated in a particular round of chain assembly). The acyltransferase (AT) domains responsible for loading of each of these building blocks onto the adjacent acyl carrier protein (ACP) domains are colour-coded accordingly.

Figure 2. Incorporation of precursors of FAS starter units into the C-26 side chains of the stambomycins.

(a) Incorporation of [U-²H]L-isoleucine 9 into the FAS starter unit 2-methylbutyryl-CoA 5, the fatty acid 6-methyloctanoic acid 13, the extender unit (4-methylhexyl)malonyl-CoA 17, and stambomycin A 1. (b) Incorporation of [U-²H]L-leucine 10 into the FAS starter unit 3-methylbutyryl-CoA 6, the fatty acid 7-methyloctanoic acid 14, the extender unit (5-methylhexyl)malonyl-CoA 18, and stambomycin B 2. (c) Incorporation of [U-²H]L-valine 11 into the FAS starter unit iso-butyryl-CoA 7, the fatty acid 6-methylheptanoic acid 15, the extender unit (4-methylpentyl)malonyl-CoA 19, and stambomycin C 3. (d) Incorporation of [U-²H]butyric acid 12 into the FAS starter unit n-butyryl-CoA 8, the fatty acid n-octanoic acid 16, the extender unit n-hexylmalonyl-CoA 20, and stambomycin D 4.

Figure 3. Discrimination between YCC and CCRC-dependent pathways using a mechanistic probe.

(a) The novel stambomycin analogue 22 is produced when heptanoic acid 21 is fed to S. ambofaciens W130. The position of incorporation of heptanoic acid into the analogue is highlighted in red. (b) Concept underlying the development of a mechanistic probe to distinguish between CCRC- and YCC-dependent pathways for unusual alkylmalonyl-CoA PKS extender unit biosynthesis. Conversion of (3-²H₂)heptanoic acid (23), via its CoA thioester 24, to pentylmalonyl-CoA (26) by a YCC-dependent pathway would result in retention of both deuterium labels, whereas one of the deuterium labels would be lost in pentylmalonyl-CoA (26) formed by a CCRC-dependent pathway, because CoA thioester 24 must undergo desaturation to 25 in order to be reductively carboxylated. (c) Route employed for the synthesis of (3-²H₂)heptanoic acid 23. (d, e) Mass spectra of stambomycin analogue 22 from LC–MS analyses of mycelial extracts of S. ambofaciens W130 cultures supplemented with unlabeled heptanoic acid (d) and (3-²H₂)heptanoic acid (23, e). The shift of one m/z unit for the doubly-charged parent ion in the right spectrum indicates that both deuterium labels are retained when (3-²H₂)heptanoic acid (23) is incorporated into stambomycin analogue 22.

Figure 4. Reveromycins produced by Streptomyces sp. SN-593 wild type and revT::samR0483 mutant.

(a) Structures of the reveromycins produced by wild type Streptomyces sp. SN-593. The site of unusual alkylmalonyl-CoA extender unit incorporation is highlighted in-red. (b, c) Mass spectra of reveromycin D (32) from UHPLC-ESI-MS analyses of ethyl acetate extracts of Streptomyces sp. SN-593 revR mutant cultures grown in the absence (b) and presence (c) of (3-²H₂)heptanoic acid (23). The increase in intensity of the m/z=674 peak in the right spectrum is consistent with incorporation of (3-²H₂)heptanoic acid (23) into reveromycin D (32) with loss of one of the two deuterium labels. (d, e, f) Extracted ion chromatograms at m/z=687–688 corresponding to [M-H]⁻ for reveromycin E from UHPLC-ESI-MS analyses of ethyl acetate extracts of wild type Streptomyces sp. SN-593 (d), the revT mutant (e) and the revT mutant (f) in which samR0483 has been expressed.

Figure 5. X-ray crystal structures of apo-MccB and holo-MccB.

(a) The hexameric overall structure of apo-MccB. (b) The substrate binding site at the subunit interface of apo-MccB. (c) Structure of MccB with 4 molecules of hexanoyl-CoA bound, showing the overall architecture of the complex with hexanoyl-CoA in space filling representation. (d) Polar contacts between hexanoyl-CoA and MccB. Residues labelled in black correspond to monomer F and residues labelled in grey correspond to monomer B of the MccB homo-dimer. (e) Residues lining the hydrophobic pocket in MccB that bind the aliphatic chain of hexanoyl-CoA. (f) Overlay of the structures of MccB (green) and PccB (cyan), highlighting residues lining the binding pockets for the aliphatic chains of hexanoyl- and propionyl-CoA, their respective substrates.

Figure 6. Bio-orthogonally-tagged stambomycin analogues produced by precursor directed biosynthesis.

(a) Azide-tagged stambomycin analogue 37 is produced when 6-azidohexanoic acid 36 is fed to S. ambofaciens W130. (b) Acetylene-tagged stambomycin analogue 39 is produced when either 6-heptynoic acid 38 or 8-nonynoic acid 40 is fed to S. ambofaciens W130. 6-heptynoic acid 38 presumably undergoes conversion to its CoA thiosester, 2-carbon elongation by the primary metabolic FAS and hydrolysis to form 8-nonynoic acid 40 before incorporation into 39.

Figure 7. Structure of primycin A.

The site of incorporation of the unusual butylmalonyl-CoA extender unit, which is proposed to be assembled by a YCC-dependent pathway, is highlighted in red.