Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin

Abstract

Mupirocin is a clinically important antibiotic produced by Pseudomonas fluorescens NCIMB 10586 that is assembled by a complex trans-AT polyketide synthase. The polyketide fragment, monic acid, is esterified by a 9-hydroxynonanoic acid (9HN) side chain which is essential for biological activity. The ester side chain assembly is initialised from a 3-hydroxypropionate (3HP) starter unit attached to the acyl carrier protein (ACP) MacpD, but the fate of this species is unknown. Herein we report the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis. These investigations reveal a complex interplay between a novel iterative or "stuttering" KS-AT didomain (MmpF), the multidomain module MmpB and multiple ACPs. This work has important implications for understanding the late-stage biosynthetic steps of mupirocin and will be important for future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol).

Keywords: Fatty Acids; Mupirocin; Natural Product Biosynthesis; Polyketide Antibiotics; Pseudomonic Acid.

Figure 1

Mupirocin biosynthesis. The C₁₇ monic acid backbone is assembled by MmpD, MmpA and MmpE. Esterification of the C₁₇ pre‐monic acid backbone (3) with the fatty acid chain (shown is the full length 9‐hydroxynonanoic acid thiol ester (2)) and tetrahydropyran ring (THP) formation yields PA‐B. PA‐B is subsequently converted to PA‐A (1) in a further multistep cascade. Accumulation of both mupiric acid and mupirocin H is observed when specific mutations in mmpD or mmpA are made and importantly also when late‐stage enzymes are inactivated. ACP—acyl carrier protein, AT—acyl transferase, ER—enoylreductase, DH—dehydratase, KR—ketoreductase, KS—ketosynthase, MT, methyl transferase, Ox—oxidoreductase, TE—thioesterase.

Figure 2

Reaction scheme and ¹H‐¹⁵N HSQC titration experiments. A) The possible fate of 3HP‐MacpD. In pathway A, MacpD delivers the 3HP starter unit to MmpF, where chain extension begins. A C₅ or C₇ carbon chain is delivered to MmpB for a final round of extension. In pathway B the 3HP unit is delivered directly to MmpB, which catalyses all three rounds of carbon chain extension, and MmpF plays no part. B) ¹H‐¹⁵N HSQC spectra of MacpD (centre), MacpD+MmpF (right) and MacpD+MmpB_KS (left).

Scheme 1

A) Conversion of 3‐hydroxyproprionate attached to MacpD to 5‐hydroxypentanoyl‐ACP. Investigations into MupE utilised crotonyl pantetheine. B) Synthetic acyl pantetheines.

Figure 3

DCC 1—Generation of 3,5‐dihydroxypentanoyl‐ACP by MmpF and MupD. A) Decarboxylative Claisen condensation (DCC) of 3HP‐MacpD by MmpF and malonyl‐MacpA/B. B) Deconvoluted spectra and corresponding Ppant ejection for malonyl‐MacpA in the assay. C) Deconvoluted spectra and corresponding Ppant ejection for the malonyl‐MacpB assay.

Scheme 2

MmpF catalyses DCC 2 and alongside tailoring enzymes (KR, DH, ER) 5‐hydroxypentanoyl‐MacpA is converted to 7‐hydroxyheptanoyl‐ACP.

Figure 4

DDC 2—MacpA and MmpF function iteratively to produce 3,7‐dihydroxyheptanoyl‐MacpA. A) Proposed reaction scheme. B) Deconvoluted spectra and corresponding Ppant ejection of 5HP‐MacpA/malonyl‐MacpA, MmpF and MupD assay. A side reaction arising presumably from transfer of malonate to MmpF and DCC with malonyl‐MacpA followed by ketoreduction led to the generation of a significant proportion of MacpA bound 3‐hydroxypentan‐1,5‐dioic acid (cyan) and was also observed in reactions using preloaded MmpF (Figure S18).

Figure 5

DCC 3—MacpA delivers a C7 chain to MmpB_KS which generates 9‐hydroxy‐3‐oxononanoyl‐ACP5. A) Proposed reaction scheme. B) Deconvoluted spectra of MacpA (left), ACP5 (centre) and the corresponding Ppant ejection (right) when 7HH‐MacpA is incubated with MmpB_KS and malonyl‐ACP5 to produce 9‐hydroxy‐3‐oxononanoyl‐ACP5. C) Deconvoluted spectra of MacpB (left), ACP5 (centre) and the corresponding Ppant ejection (right) when 7HH‐MacpB is incubated with MmpB_KS and malonyl‐ACP5. Key: apo ACP (grey), holo ACP (pink), malonyl‐ACP (blue), 7HH‐ACP (purple) and 9‐hydroxy‐3‐oxononanoyl‐ACP5 (red).

Figure 6

MmpF is a fully reducing stuttering module that controls FAS within mupirocin biosynthesis. A) MmpF catalyses two extensions of the 3HP‐MacpD starter unit, alongside trans‐acting tailoring enzymes, before MmpB exclusively mediates the extension of a C₇ acyl chain to the 9‐hydroxynonanoic fatty acid. B) Thiomarinol A and metabolites produced by a ΔtmlU (CoA ligase) mutant. C) Metabolites from ΔmupW and ΔmmpEOR/ΔmupW mutants (MmpEOR is the oxidoreductase domain in MmpE responsible for epoxide formation).

Figure 7

Different pathways towards the coupling of fatty acid synthesis and esterification. Pathway A) 3HP‐MacpD is presumed to be esterified to a pre‐monic acid moiety before fatty acid synthesis. B) Alternative pathway proposed within this study.