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Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa

View Article: PubMed Central - PubMed

ABSTRACT

Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.

No MeSH data available.


Kdo2-Lipid A biosynthetic pathway in E. coli3.Kdo2-Lipid A synthesis from UDP-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by nine enzymes. The fourth step of the pathway involves the hydrolysis of a pyrophosphate moiety of UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) and yields UMP and 2,3-diacylglucosamine-1-phosphate (lipid X). The enzyme responsible for this hydrolysis reaction is the specific pyrophosphatase LpxH. In a few bacteria, this reaction is catalyzed by LpxI4. In the fifth step, LpxB condenses UDP-2,3-diacyl-GlcN with lipid X to form 2′,3′-diacylglucosamine-(β,1′-6)-2,3-diacylglucosamine 1-phosphate (DSMP).
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f1: Kdo2-Lipid A biosynthetic pathway in E. coli3.Kdo2-Lipid A synthesis from UDP-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by nine enzymes. The fourth step of the pathway involves the hydrolysis of a pyrophosphate moiety of UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) and yields UMP and 2,3-diacylglucosamine-1-phosphate (lipid X). The enzyme responsible for this hydrolysis reaction is the specific pyrophosphatase LpxH. In a few bacteria, this reaction is catalyzed by LpxI4. In the fifth step, LpxB condenses UDP-2,3-diacyl-GlcN with lipid X to form 2′,3′-diacylglucosamine-(β,1′-6)-2,3-diacylglucosamine 1-phosphate (DSMP).

Mentions: In Escherichia coli lipid A is synthesized from UDP-N-acetylglucosamine (UDP-GlcNAc) in nine steps, each of which are catalyzed by different enzymes (Fig. 1)3. Most of these constitutive enzymes are well conserved in gram-negative bacteria. In the fourth step in this pathway, UDP-2,3-diacylglucosamine is hydrolyzed to 2,3-diacylglucosamine-1-phosphate (also called lipid X) and UMP. This step is catalyzed by the specific pyrophosphatase LpxH in 70% of gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, and by LpxI in the remaining 30%345. Although these two enzymes provide the same product from the same substrate, they have no sequence similarity and are not found in the same organisms. Furthermore, they have different metal ion requirements and attack different phosphorus atoms during hydrolysis; LpxH attacks the α-phosphate of UDP-2,3-diacylglucosamine, and LpxI attacks the β-phosphate4. In spite of these differences, LpxI can compensate for LpxH loss in E. coli; thus, the two enzymes are considered to have the same role in the context of lipid A biosynthesis4.


Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa
Kdo2-Lipid A biosynthetic pathway in E. coli3.Kdo2-Lipid A synthesis from UDP-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by nine enzymes. The fourth step of the pathway involves the hydrolysis of a pyrophosphate moiety of UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) and yields UMP and 2,3-diacylglucosamine-1-phosphate (lipid X). The enzyme responsible for this hydrolysis reaction is the specific pyrophosphatase LpxH. In a few bacteria, this reaction is catalyzed by LpxI4. In the fifth step, LpxB condenses UDP-2,3-diacyl-GlcN with lipid X to form 2′,3′-diacylglucosamine-(β,1′-6)-2,3-diacylglucosamine 1-phosphate (DSMP).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5016852&req=5

f1: Kdo2-Lipid A biosynthetic pathway in E. coli3.Kdo2-Lipid A synthesis from UDP-N-acetylglucosamine (UDP-GlcNAc) is catalyzed by nine enzymes. The fourth step of the pathway involves the hydrolysis of a pyrophosphate moiety of UDP-2,3-diacylglucosamine (UDP-2,3-diacyl-GlcN) and yields UMP and 2,3-diacylglucosamine-1-phosphate (lipid X). The enzyme responsible for this hydrolysis reaction is the specific pyrophosphatase LpxH. In a few bacteria, this reaction is catalyzed by LpxI4. In the fifth step, LpxB condenses UDP-2,3-diacyl-GlcN with lipid X to form 2′,3′-diacylglucosamine-(β,1′-6)-2,3-diacylglucosamine 1-phosphate (DSMP).
Mentions: In Escherichia coli lipid A is synthesized from UDP-N-acetylglucosamine (UDP-GlcNAc) in nine steps, each of which are catalyzed by different enzymes (Fig. 1)3. Most of these constitutive enzymes are well conserved in gram-negative bacteria. In the fourth step in this pathway, UDP-2,3-diacylglucosamine is hydrolyzed to 2,3-diacylglucosamine-1-phosphate (also called lipid X) and UMP. This step is catalyzed by the specific pyrophosphatase LpxH in 70% of gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, and by LpxI in the remaining 30%345. Although these two enzymes provide the same product from the same substrate, they have no sequence similarity and are not found in the same organisms. Furthermore, they have different metal ion requirements and attack different phosphorus atoms during hydrolysis; LpxH attacks the α-phosphate of UDP-2,3-diacylglucosamine, and LpxI attacks the β-phosphate4. In spite of these differences, LpxI can compensate for LpxH loss in E. coli; thus, the two enzymes are considered to have the same role in the context of lipid A biosynthesis4.

View Article: PubMed Central - PubMed

ABSTRACT

Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.

No MeSH data available.