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A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis
Paul D Walker1,2, Matthew T Rowe1, Ashley J Winter1
1School of Chemistry , University of Bristol , Cantock's Close , Bristol , BS8 1TS , United Kingdom.
Abstract:
Mupirocin, a commercially available antibiotic produced by Pseudomonas fluorescens NCIMB 10586, and thiomarinol, isolated from the marine bacterium Pseudoalteromonas sp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties in mup and tml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively, via the MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternate trans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein-protein interactions for this region of the ACP.
Insights
Researchers elucidated the formation of fatty acid moieties in mupirocin and thiomarinol antibiotics. They discovered specific enzymes convert malonyl coenzyme A and succinyl CoA into starter units, clarifying polyketide biosynthesis pathways.
Area of Science:
- Biochemistry and Molecular Biology
- Natural Product Biosynthesis
- Enzymology
Background:
- Mupirocin and thiomarinol are antibiotics derived from polyketide pathways.
- The biosynthesis of their unique fatty acid components (9-hydroxynonanoic acid and 8-hydroxyoctanoic acid) remains unclear.
- Understanding starter unit generation is crucial for elucidating these complex biosynthetic mechanisms.
Purpose of the Study:
- To define the mechanisms of 9-hydroxynonanoic acid and 8-hydroxyoctanoic acid starter unit formation.
- To investigate the roles and selectivities of key enzymes in the mupirocin and thiomarinol pathways.
- To characterize the structural and functional significance of acyl carrier proteins (ACPs) involved.
Main Methods:
- Expression and purification of mupirocin (MupQ, MupS, MacpD) and thiomarinol (TmlQ, TmlS, TacpD) pathway proteins.
- Enzymatic assays using malonyl coenzyme A (CoA) and succinyl CoA to identify starter unit products (3-hydroxypropionoyl and 4-hydroxybutyryl).
- Mix-and-match experiments to assess enzyme and ACP selectivity.
- Nuclear Magnetic Resonance (NMR) spectroscopy to determine the structure of MacpD.
Main Results:
- MupQ/TmlQ and MupS/TmlS enzymes catalyze the formation of 3-hydroxypropionoyl and 4-hydroxybutyryl starter units, respectively, via an unusual bis-CoA/ACP thioester intermediate.
- MupQ/TmlQ exhibit high selectivity for their cognate CoA substrates.
- MacpD/TacpD are interchangeable, but alternative ACPs are non-functional; MupS/TmlS show varied selectivity for substrates and ACPs.
- The C-terminal extension of MacpD, important for mupirocin titers, is not essential for initial 3-HP generation but may play roles in downstream protein interactions.
Conclusions:
- The study elucidates the enzymatic pathway for generating starter units in mupirocin and thiomarinol biosynthesis.
- Enzyme and ACP selectivity plays a critical role in pathway fidelity.
- The C-terminal extension of MacpD has implications beyond initial substrate processing, potentially influencing protein complex formation.
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