Mupirocin
Pseudomonic acid A:
Chemical formula: C26H44O Molecular Weight 500.622
CAS #: 12650-69-0
IUPAC name: 9-[(E)-4-[(2S,3R,4R,5S)-3,4-dihydroxy-5-[[(2S,3S)-3- [(2S,3S)-3-hydroxybutan-2-yl]oxiran-2-yl]methyl] oxan-2-yl]-3-methylbut-2-enoyl]oxynonanoic acid
Introduction
Mupirocin is an antibiotic isolated from Pseudomonas fluorescens NCIMB 10586 that is effective against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus [1],[2]. Mupirocin is a mixture of several pseudomonic acids (Figure 1), with pseudomonic acid A (PA-A) constituting greater than 90% of the mixture. Also present in mupirocin are pseudomonic acid B with and additional hydroxyl group at C8 [3], pseudomonic acid C with a double bond between C10 and C11, instead of the epoxide of PA-A [4], and pseudomonic acid D with a double bond at C4` and C5` in the 9-hydroxynonanoic acid portion of mupirocin [5].
Figure 1. Mupirocin is primarily composed of pseudomonic acid A with minor constituents psuedomonic acids B, C, and D. Pseudomonic acid A-D are shown as structures A-D, respectively.
Mechanism of Action
Mupirocin has been shown to strongly inhibit protein and RNA synthesis in Staphylococcus aureus while DNA and cell wall formation were also negatively impacted but to a lesser degree [6]. The inhibition of RNA synthesis was shown to be in response to a lack of one amino acid, isoleucine [7]. In vivo studies in Escherichia coli demonstrated that pseudomonic acid inhibits isoleucine t-RNA synthetase (IleRS) [2]. This mechanism of action is shared with furanomycin, an analog of isoleucine [8].
Resistance to Mupirocin
Shortly after the clinical use of Mupirocin began, strains of Staphylococcus aureus that were resistant to mupirocin emerged [9]. Two distinct populations of mupirocin-resistant S. aureus were isolated. One strain posessed low-level resistance, MuL, (MIC = 8-256 mg/L) and another posessed high-level resistance, MuH, (MIC > 256 mg/L) [9]. Resistance in the MuL strains is probably due to mutations in the organism’s wild-type isoleucinyl-tRNA synthetase. In E. coli IleRS, a single amino acid mutation was shown to alter mupirocin resistance [10]. MuH is linked to the acquisition of a separate Ile synthetase gene, mupA [11]. Mupirocin is not a viable antibiotic against MuH strains. Other antibiotic agents such as azelaic acid, nitrofurazone, silver sulphadiazine, and ramoplanin have been shown to be effective against MuH strains [9].
The mechanism of mupirocin differs from other clinical antibiotics rendering cross-resistance to other antibiotics unlikely [9]. However, the MupA gene may co-transfer with other antibacterial resistance genes. This has been observed already with resistance genes for triclosan, tetracycline, and trimethoprim [9].
Biosynthesis of Pseudomonic Acid A
The 74 kb mupirocin cluster contains six multi-domain enzymes and twenty six other peptides (Table 1) [12]. Four large multi-domain type I polyketide synthase (PKS) proteins are encoded, as well as several single function enzymes with sequence similarity to type II PKSs [12]. It is therefore believed that mupirocin is constructed by a mixed type I and type II PKS system. The mupirocin cluster exhibits an atypical acyltransferase (AT) organization, in that there are only two AT domains and both are found on the same protein, MmpC. These AT domains are the only domains present on MmpC, while the other three type I PKS proteins contain no AT domains [12]. The mupirocin pathway also contains several tandem ACP doublets or triplets. This may be an adaptation to increase the throughput rate or to bind multiple substrates simultaneously [12].
Pseudomonic acid A is the product of an esterification between the 17C polyketide monic acid and the 9C fatty acid 9-hydroxynonanoic acid. The possibility that the entire molecule is assembled as a single polyketide with a Baeyer-Villiger oxidation inserting an oxygen into the carbon backbone has been ruled out because C1 of monic acid and C9’ of 9-Hydroxynonanoic acid are both derived from C1 of acetate [13].
Table 1. The gene cluster for the biosynthesis of mupirocin has been described. ACP=acyl carrier protein, AT=acyl transferase, DH=dehydratase, ER=enoyl reductase, FMNH2=Flavin mononucleotide (reduced), HMG=3-hydroxy-3-methylglutaric acid, MeT=methyl transferase, NADH=Nicotinamide adenine dinucleotide (reduced), NADPH=Nicotinamide adenine dinucleotide phosphate (reduced), N-AHL=N-acyl homoserine lactone, KR=ketoreductase, KS=ketosynthase, TE=thioesterase.
Monic acid (MA) biosynthesis
Biosynthesis of the 17C monic acid unit begins on MmpD (Figure 2) [12]. One of the AT domains from MmpC may transfer an activated acetyl group from acetyl-Coenzyme A (CoA) to the first ACP domain. The chain is extended by malonyl-CoA, followed by a SAM-dependant methylation at C12 and reduction of the B-keto group to an alcohol. The dehydration (DH) domain in module 1 is predicted to be non-functional due to a mutation in the conserved active site region. Module 2 adds another two carbons by malonyl-CoA extender unit, followed by ketoreduction (KR) and dehydration. Module three adds a malonyl-CoA extender unit, followed by SAM-dependant methylation at C8, ketoreduction, and dehydration. Module 4 extends the molecule with a malonyl-CoA unit followed by ketoreduction.
Figure 2. The proposed biosynthetic route of monic acid. The biosynthesis of monic acid is not colinear but has been rearranged in this diagram. The protein name is displayed inside of the arrow with module and domain structure listed below. ACP=acyl carrier protein, AT=acyl transferase, DH=dehydratase, ER=enoyl reductase, HMG=3-hydroxy-3-methylglutaric acid, MeT=methyl transferase, KR=ketoreductase, KS=ketosynthase, TE=thioesterase.
Assembly of monic acid is continued by the transfer of the 12C product of MmpD to MmpA [12]. Two more rounds of extension with malonyl-CoA units are achieved by module 5 and 6. Module 5 also contains a KR domain.
The keto group at C3 is replaced with a methyl group in a multi-step reaction (Figure 3). MupG begins by decarboxylating a malonyl-ACP. The alpha carbon of the resulting acetyl-ACP is linked to C3 of the polyketide chain by MupH. This intermediate is dehydrated and decarboxylated by MupJ and MupK, respectively [12].
Figure 3. The C15 methyl group of monic acid is attached to C3 by the following reaction scheme. MupH is an 3-hydroxy-3-methylglutarate-Coenzyme A synthase, MupJ and MupK are Enoyl-CoA hydratases [12].
The formation of the furan ring requires many enzyme mediated steps (Figure 4). The double bond between C8 and C9 is proposed to migrate to between C8 and C16 [14]. Gene knock-out experiments of mupO, mupU, mupV, and macpE have eliminated PA-A production [14]. PA-B production is not removed by these knockouts, demonstrating that PA-B is not created by hydroxylating PA-A. A knock-out of mupW eliminated the furan ring, identifying MupW as being involved in ring formation [14]. It is not known if this occurs before or after the esterification of monic acid to 9-hydroxynonanoic acid.
Figure 4. The pyran ring of mupirocin is generated in this proposed multistep reaction [15]. Gene knockouts of mupO, mupU, mupV and macpE abolish PA-A production but not PA-B production, demonstrating that PA-B is a precusor to PA-A [14].
The epoxide of PA-A at C10-11 is believed to be inserted after pyran formation by a cytochrome P450 such as MupO [12]. A gene knockout of mupO abolished PA-A production but PA-B, which also conatins the C10-C11 epoxide, remained [14]. This indicates that MupO is either not involved or is not essential for this epoxidation step.
9-hydroxy-nonanoic acid (9-HN) biosynthesis
The nine carbon fatty acid 9-hydroxy-nonanoic acid is derived as a separate compound and later esterified to monic acid to form pseudomonic acid. 13C labeled acetate feeding has shown that C1-C6 are constructed with acetate in the canonical fashion of fatty acid synthesis. C7’ shows only C1 labeling of acetate while C8’ and C9’ show a reversed pattern of 13C labeled acetate [13]. It is speculated that C7-9 arises from a 3-hydroxypropionate starter unit, which is extended three times with malonyl-CoA and fully reduced to yield 9-HN. It has also been suggested that 9-HN is initiated by 3-hydroxy-3-methylglutaric acid (HMG). This latter theory was not supported by feeding of [3-14C] or [3,6-13C2]-HMG [16].
MmpB is proposed to catalyze the synthesis of 9-HN (Figure 5). MmpB contains a KS, KR, DH, 3 ACPs, and a thioesterase (TE) domain [12]. It does not contain an enoyl reductase (ER) domain which would be required for the complete reduction to the nine carbon fatty acid. MupE is a single domain protein that shows sequence similarity to known ER domains and may complete the reaction [12]. It also remains possible that 9-hydroxynonanoic acid is derived partially or entirely from outside of the mupirocin cluster.
Figure 5. MmpB is proposed to synthesize 9-HN with a 3-hydroxy-propionate starter unit and three malonyl-CoA extenter units. The domain structure of MmpB is shown below along side with MupE, the proposed enoyl reductase required for complete saturation of 9-HN. ACP=acyl carrier protein, DH=dehydratase, ER=enoyl reductase, KR=ketoreductase, KS=ketosynthase, TE=thioesterase.
References:
1. Fuller, A.T., et al., Pseudomonic Acid: an Antibiotic produced by Pseudomonas fluorescens. Nature, 1971. 234(5329): p. 416-417.
2. Hughes, J. and G. Mellows, Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid. 1978, Portland Press Ltd. p. 305.
3. Chain, E.B. and G. Mellows, Pseudomonic acid. Part 3. The structure of Pseudomonic acid BJ Chem. Soc. 1977. p. 318-322.
4. Clayton, J.P., P.J. Ohanlon, and N.H. Rogers, Structure and Configuration of Pseudomonic Acid-C. Tetrahedron Letters, 1980. 21(9): p. 881-884.
5. Ohanlon, P.J., N.H. Rogers, and J.W. Tyler, The Chemistry of Pseudomonic Acid .6. Structure and Preparation of Pseudomonic Acid-D. Journal of the Chemical Society-Perkin Transactions 1, 1983(11): p. 2655-2657.
6. Hughes, J. and G. Mellows, On the mode of action of pseudomonic acid: inhibition of protein synthesis in Staphylococcus aureus. 1978, Journal@rchive. p. 330-335.
7. Haseltin.Wa and R. Block, Synthesis of Guanosine Tetraphosphate and Pentaphosphate Requires Presence of a Codon-Specific, Uncharged Transfer Ribonucleic-Acid in Acceptor Site of Ribosomes - (Stringent Control Ppgpp (Msi) and Pppgpp (Msii) Protein Synthesis Escherichia-Coli). Proceedings of the National Academy of Sciences of the United States of America, 1973. 70(5): p. 1564-1568.
8. Tanaka, K., M. Tamaki, and S. Watanabe, Effect of furanomycin on the synthesis of isoleucyl-tRNA. 1969. p. 244-5.
9. Cookson, B.D., The emergence of mupirocin resistance: a challenge to infection control and antibiotic prescribing practice. 1998, Br Soc Antimicrob Chemo. p. 11-18.
10. Yanagisawa, T., et al., Relationship of Protein-Structure of Isoleucyl-Transfer-Rna Synthetase with Pseudomonic Acid Resistance of Escherichia-Coli - Proposed Mode of Action of Pseudomonic Acid as an Inhibitor of Isoleucyl-Transfer-Rna Synthetase. Journal of Biological Chemistry, 1994. 269(39): p. 24304-24309.
11. Gilbart, J., C.R. Perry, and B. Slocombe, High-Level Mupirocin Resistance in Staphylococcus-Aureus - Evidence for 2 Distinct Isoleucyl-Transfer Rna-Synthetases. Antimicrobial Agents and Chemotherapy, 1993. 37(1): p. 32-38.
12. El-Sayed, A.K., et al., Characterization of the Mupirocin Biosynthesis Gene Cluster from Pseudomonas fluorescens NCIMB 10586. 2003, Elsevier. p. 419-430.
13. Feline, T.C., et al., Pseudomonic acid. Part 2. Biosynthesis of pseudomonic acid A. 1977, Royal Society of Chemistry. p. 309-318.
14. Cooper, S.M., et al., Shift to Pseudomonic Acid B Production in P. fluorescens NCIMB10586 by Mutation of Mupirocin Tailoring Genes mupO, mupU, mupV, and macpE. 2005, Elsevier. p. 825-833.
15. Hothersall, J., et al., Mutational Analysis Reveals That All Tailoring Region Genes Are Required for Production of Polyketide Antibiotic Mupirocin by Pseudomonas fluorescens: PSEUDOMONIC ACID B BIOSYNTHESIS PRECEDES PSEUDOMONIC ACID A. 2007, ASBMB. p. 15451.
16. Martin, F.M. and T.J. Simpson, Biosynthetic-Studies on Pseudomonic Acid (Mupirocin), a Novel Antibiotic Metabolite of Pseudomonas-Fluorescens. Journal of the Chemical Society-Perkin Transactions 1, 1989(1): p. 207-209.
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