WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.
The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.
Mamat
Uwe
U
Divisions of Structural Biochemistry, D-23845 Borstel, Germany.
Schmidt
Helgo
H
Divisions of Structural Biochemistry, D-23845 Borstel, Germany; Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, D-23538 Lübeck, Germany.
Munoz
Eva
E
the Institutes of Chemistry, D-23538 Lübeck, Germany.
Lindner
Buko
B
Immunochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, D-23845 Borstel, Germany.
Fukase
Koichi
K
Department of Chemistry, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Hanuszkiewicz
Anna
A
Divisions of Structural Biochemistry, D-23845 Borstel, Germany.
Wu
Jing
J
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
Meredith
Timothy C
TC
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
Woodard
Ronald W
RW
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
Hilgenfeld
Rolf
R
Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, D-23538 Lübeck, Germany.
Mesters
Jeroen R
JR
Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, D-23538 Lübeck, Germany.
Holst
Otto
O
Divisions of Structural Biochemistry, D-23845 Borstel, Germany.
D001419:Bacteria / Q000378:metabolism*; D002241:Carbohydrates / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry; D008954:Models, Biological; D009713:Nucleotidyltransferases / Q000378:metabolism; D012475:Salmonella / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D013696:Temperature; D014166:Transferases / Q000737:chemistry* / Q000502:physiology*
D002241:Carbohydrates; D029968:Escherichia coli Proteins; D008055:Lipids; D008070:Lipopolysaccharides; D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase