Subsystem: Lipid A-Ara4N pathway ( Polymyxin resistance )

This subsystem's description is:

Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin.
The enzymes responsible for the synthesis of Ara4N precursors and its transfer to lipid A are encoded in the pmrHFIJKLM operon and the pmrE gene (pmr = Polymyxin resistance) (Gunn et al., 1998). These proteins are highly conserved in gram-negative bacteria such as E. coli and the human pathogens Pseudomonas aeruginosa, Burkolderia cepacia, Yersinia pestis, and all types of Salmonella (Ref.2).

=========== VARIANTS:===================================

1.0 - full set of Polymyxin resistance genes: pmrHFIJKLM operon + Ugd(pmrE gene)
2.0 - pmrHFIJKLM operon, no Ugd(pmrE gene)- UDP-glucose dehydrogenase (EC 1.1.1.22)
x -stands for the missing genes or incomplete pathway

=============INTRODUCTION:===================================

For more information, please check out the description and the additional notes tabs, below

Literature ReferencesLipopolysaccharide endotoxins. Raetz CR Annual review of biochemistry 200212045108
DiagramFunctional RolesSubsystem SpreadsheetDescriptionAdditional Notes 

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Taxonomy Pattern 
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Domain
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UgdArnA_DHArnA_FTArnBArnCArnTPmrJPmrLPmrDPmrMPmrG
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Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa can modify the structure of lipid A in their outer membrane with 4-amino-4-deoxy-l-arabinose (Ara4N). Such modification results in resistance to cationic antimicrobial peptides of the innate immune system and antibiotics such as polymyxin.
The enzymes responsible for the synthesis of Ara4N precursors and its transfer to lipid A are encoded in the pmrHFIJKLM operon and the pmrE gene (pmr = Polymyxin resistance) (Gunn et al., 1998). These proteins are highly conserved in gram-negative bacteria such as E. coli and the human pathogens Pseudomonas aeruginosa, Burkolderia cepacia, Yersinia pestis, and all types of Salmonella (Ref.2).

=========== VARIANTS:===================================

1.0 - full set of Polymyxin resistance genes: pmrHFIJKLM operon + Ugd(pmrE gene)
2.0 - pmrHFIJKLM operon, no Ugd(pmrE gene)- UDP-glucose dehydrogenase (EC 1.1.1.22)
x -stands for the missing genes or incomplete pathway

=============INTRODUCTION:===================================
Nonpolar mutations of the individual proteins in the operon showed that all proteins, except pmrM, are essential for the addition of Ara4N to lipid A and polymyxin resistance, making them excellent potential targets for drug design.
In polymyxin-resistant E. coli and S. typhimurium, biosynthesis of the L-Ara4N moiety begins with oxidation of UDP-glucose to UDP-GlcUA. Next, the C-terminal domain of ArnA catalyzes the NAD+-dependent oxidative decarboxylation of UDP-GlcUA to yield UDP-4''-ketopentose, which is converted to UDP-L-Ara4N by ArnB. The N-terminal domain of ArnA uses N-10-formyltetrahydrofolate to convert UDP-L-Ara4N to UDP-L-Ara4FN. ArnC (a distant ortholog of dolichyl phosphate-mannose synthase) selectively transfers the L-Ara4FN residue of UDP-{beta}-L-Ara4FN to undecaprenyl phosphate, forming undecaprenyl phosphate-{alpha}-L-Ara4FN. The ArnD-dependent deformylation of this substance to undecaprenyl phosphate-{alpha}-L-Ara4N (which accumulates in polymyxin resistant mutants) likely occurs on the inner leaflet of the inner membrane and may prevent reversal of the ArnC reaction. After transport to the outer surface of the inner membrane, the membrane protein ArnT transfers the L-Ara4N moiety to lipid A.

======= Functional Roles:===================

- Ugd - UDP-glucose dehydrogenase (EC 1.1.1.22)
Conversion of UDP-Glucose into UDP-Glucuronic acid (UDP-GlcA) is catalyzed by the product of the pmrE gene, a well-characterized dehydrogenase

- ArnA (pmrI)- Polymyxin resistance bifunctional protein ArnA, formyltransferase/decarboxylase.
ArnA is a key enzyme in the lipid A modification pathway, and its deletion abolishes both the Ara4N-lipid A modification and polymyxin resistance.
ArnA is a bifunctional enzyme. The N-terminal 300 amino acids encode the transformylase activity, whereas the C-terminal 360 amino acids are responsible for the dehydrogenase activity. ArnA can catalyze (i) the NAD(+)-dependent decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose and (ii) the N-10-formyltetrahydrofolate-dependent formylation of UDP-4-amino-4-deoxy-l-arabinose. It is shown that the NAD(+)-dependent decarboxylating activity is contained in the 360 amino acid C-terminal domain of ArnA. This domain is separable from the N-terminal fragment, and its activity is identical to that of the full-length enzyme. The crystal structure of the ArnA decarboxylase domain from E. coli confirms that the enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family.
Both the transformylase and dehydrogenase activities catalyzed by ArnA are required for lipid A modification with Ara4N and polymyxin resistance (Breazeale et al., 2005). These activities can be physically separated by expressing the individual ArnA domains ( Ref. 2, 3 ).


- ArnB (pmrH) - UDP-4-amino-4-deoxy-L-arabinose--oxoglutarate aminotransferase (EC 2.6.1.-); Polymyxin resistance protein ArnB.
Lipid A modification with 4-amino-4-deoxy-L-arabinose; step 3. Catalyzes the conversion of UDP-4-keto-arabinose (UDP-Ara4O) to UDP-4-amino-4-deoxy-L-arabinose (UDP-L-Ara4N). The modified arabinose is attached to lipid A and is required for resistance to polymyxin and cationic antimicrobial peptides (Ref. 4).
Reaction:
UDP-4-amino-4-deoxy-L-arabinose + 2-oxoglutarate = UDP-beta-(L-threo-pentapyranosyl-4''-ulose) + glutamate.

- ArnC ((pmrF) - Polymyxin resistance protein ArnC, glycosyl transferase (EC 2.4.-.-) - (a distant ortholog of dolichyl phosphate-mannose synthase) selectively transfers the L-Ara4FN residue of UDP-{beta}-L-Ara4FN (magenta rectangle) to undecaprenyl phosphate, forming undecaprenyl phosphate-{alpha}-L-Ara4FN.

- ArnD - the ArnD-dependent deformylation of this substance to undecaprenyl phosphate-{alpha}-L-Ara4N (which accumulates in polymyxin resistant mutants)) likely occurs on the inner leaflet of the inner membrane and may prevent reversal of the ArnC reaction. After transport to the outer surface of the inner membrane, the membrane protein ArnT transfers the L-Ara4N moiety to lipid A.

- ArnT (pmrK)- Polymyxin resistance protein ArnT, undecaprenyl phosphate-alpha-L-Ara4N transferase (Melittin resistance protein pqaB) - Catalyzes the transfer of the L-Ara4N moiety of the glycolipid undecaprenyl phosphate-alpha-L-Ara4N to lipid A. The modified arabinose is attached to lipid A and is required for resistance to polymyxin and cationic antimicrobial peptides.

Additional enzymes in the pmrHFIJKLM operon have been proposed to deformylate undecaprenyl-P-Ara4FN and complete the transfer of Ara4N to lipid A. However, their activity has not yet been demonstrated in vitro.

=============REFERENCES:========================

1. Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002;71:635-700. Epub 2001 Nov 9. Review.PMID: 12045108

2. Gatzeva-Topalova PZ, May AP, Sousa MC. Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance.
Structure (Camb). 2005 Jun;13(6):929-42.

3. Breazeale et al., 2005 S.D. Breazeale, A.A. Ribeiro, A.L. McClerren and C.R.H. Raetz, A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-Amino-4-deoxy-L-arabinose. Identification and function of UDP-4-deoxy-4-formamido-L-arabinose, J. Biol. Chem. 280 (2005), pp. 1415414167.

4. Breazeale SD; Ribeiro AA; Raetz CRH. Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose. 2003, J. Biol. Chem., 278, 24731-24739