The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases

Xiang Ruan, David E. Loyola, Cristina L. Marolda, José M. Perez-Donoso, Miguel A. Valvano

Resultado de la investigación: Article

27 Citas (Scopus)

Resumen

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5′-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X 9GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

Idioma originalEnglish
Páginas (desde-hasta)288-299
Número de páginas12
PublicaciónGlycobiology
Volumen22
N.º2
DOI
EstadoPublished - 1 feb 2012

Huella dactilar

O Antigens
Glycosyltransferases
Ligases
Metal ions
Lipopolysaccharides
Arginine
Metals
Ions
Histidine
Sugars
Lipid A
Diphosphates
Membranes
Oligosaccharides
Computer Simulation
Adenosine
Escherichia coli
Adenosine Triphosphate
Aspartic Acid
Alanine

ASJC Scopus subject areas

  • Biochemistry

Citar esto

Ruan, Xiang ; Loyola, David E. ; Marolda, Cristina L. ; Perez-Donoso, José M. ; Valvano, Miguel A. / The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases. En: Glycobiology. 2012 ; Vol. 22, N.º 2. pp. 288-299.
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title = "The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases",
abstract = "WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5′-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X 9GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.",
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The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases. / Ruan, Xiang; Loyola, David E.; Marolda, Cristina L.; Perez-Donoso, José M.; Valvano, Miguel A.

En: Glycobiology, Vol. 22, N.º 2, 01.02.2012, p. 288-299.

Resultado de la investigación: Article

TY - JOUR

T1 - The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases

AU - Ruan, Xiang

AU - Loyola, David E.

AU - Marolda, Cristina L.

AU - Perez-Donoso, José M.

AU - Valvano, Miguel A.

PY - 2012/2/1

Y1 - 2012/2/1

N2 - WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5′-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X 9GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

AB - WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5′-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X 9GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

KW - glycosyltransferase

KW - lipopolysaccharide

KW - membrane protein

KW - O-antigen

KW - oligosaccharyltransferase

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DO - 10.1093/glycob/cwr150

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