Publications

• The structures of oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant Escherichia coli strain expressing the gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] of Chlamydia psittaci 6BC.

  Holst O., Bock K., Brade L., Brade H.

  The lipopolysaccharide from the recombinant strain Escherichia coli F515-140 containing the cloned gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] from Chlamydia psittaci 6BC was isolated and sequentially de-O-acylated and de-N-acylated. The products were separated by high-per ... >> More

  The lipopolysaccharide from the recombinant strain Escherichia coli F515-140 containing the cloned gene gseA [3-deoxy-D-manno-octulopyranosonic acid (Kdo) transferase] from Chlamydia psittaci 6BC was isolated and sequentially de-O-acylated and de-N-acylated. The products were separated by high-performance anion-exchange chromatography into three fractions, two of which contained a single compound. Their structures were elucidated by high-field NMR spectroscopy as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 1) (tetrasaccharide bisphosphate) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. & Brade, H. (1993) Eur. J. Biochem. 214, 703-710] and alpha-Kdo-(2-->4)-[alpha-Kdo-(2-->8)-]-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 4) (hexasaccharide bisphosphate). The third fraction comprised two pentasaccharide bisphosphates, which could be separated by affinity chromatography using an immobilized monoclonal antibody specific for the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. The bound fraction was identified as alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6)-alpha-D-GlcN 1,4'-P2 (compound 2) [Holst, O., Broer, W., Thomas-Oates, J. E., Mamat, U. & Brade, H. (1993) Eur. J. Biochem. 214, 703-710], whereas the unbound fraction was identified as alpha-Kdo-(2-->4)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->6)-beta-D-GlcN-(1-->6 )-alpha-D-GlcN 1,4'-P2 (compound 3). This novel Kdo tetrasaccharide extends our knowledge on multifunctional Kdo transferases. << Less

  Eur. J. Biochem. 229:194-200(1995) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3-deoxy-alpha-D-manno-octulosonic acid transferase of Chlamydia pneumoniae strain TW-183.

  Loebau S., Mamat U., Brabetz W., Brade H.

  The gene kdtA of Chlamydia pneumoniae strain TW-183, encoding the enzyme 3-deoxy-alpha-D-manno-octulosonic acid (Kdo) transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 6 ... >> More

  The gene kdtA of Chlamydia pneumoniae strain TW-183, encoding the enzyme 3-deoxy-alpha-D-manno-octulosonic acid (Kdo) transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 69% similarity and 43% identity with KdtA of Chlamydia trachomatis and Chlamydia psittaci. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and the primary gene product was characterized as a multifunctional glycosyltransferase. Cell-free extracts generated in vitro the genus-specific epitope of Chlamydia composed of the trisaccharide alphaKdo(2-8)alphaKdo(2-4)alphaKdo. The results show that a single polypeptide affords three different glycosidic bonds, which is in contradiction to the dogma of glycobiology: 'one enzyme - one glycosidic bond'. << Less

  Mol. Microbiol. 18:391-399(1995) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and Kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli.

  Brabetz W., Mueller-Loennies S., Brade H.

  The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E ... >> More

  The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, and its nucleotide sequence was identical to H. influenzae DSM11121. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and characterized in vitro to encode a monofunctional Kdo transferase. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated at position 4. The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS. Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structures synthesized by H. influenzae I69. << Less

  J. Biol. Chem. 275:34954-34962(2000) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.

  Mamat U., Schmidt H., Munoz E., Lindner B., Fukase K., Hanuszkiewicz A., Wu J., Meredith T.C., Woodard R.W., Hilgenfeld R., Mesters J.R., Holst O.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 284:22248-22262(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli.

  Belunis C.J., Raetz C.R.

  The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that o ... >> More

  The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli. << Less

  J. Biol. Chem. 267:9988-9997(1992) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A mono-functional 3-deoxy-D-manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influenzae.

  White K.A., Kaltashov I.A., Cotter R.J., Raetz C.R.

  Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. ... >> More

  Lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue, linked to the 6' position of lipid A. In Escherichia coli and related organisms, a Kdo disaccharide is attached to lipid A. In previous studies, we cloned the gene (kdtA) encoding the E. coli Kdo transferase and demonstrated that homogeneous preparations of KdtA polypeptide catalyzed the attachment of both Kdo groups to the precursor, lipid IVA. E. coli KdtA produced only traces of mono-glycosylated product. We now show that a single Kdo is transferred to lipid IVA in extracts of H. influenzae. The mono-functional Kdo transferase of H. influenzae is membrane-bound, and the reaction is dependent upon a CMP-Kdo-generating system, as in E. coli. The specific activity of Kdo transfer to lipid IVA is 0.5-1 nmol/min/mg in H. influenzae membranes. Utilizing solubilized H. influenzae membranes, milligram quantities of Kdo-lipid IVA were prepared for analysis. Matrix-assisted laser desorption/ionization mass spectrometry revealed a parent ion (M - H)-at m/z 1626.0, consistent with the addition of a single Kdo moiety. Like lipid IVA, Kdo-lipid IVA was an excellent substrate for the bi-functional Kdo transferase of E. coli. In membranes of H. influenzae, but not E. coli, Kdo-lipid IVA was further phosphorylated in the presence of ATP, yielding a mono-phosphorylated Kdo-lipid IVA with a parent ion (M - H)-at m/z 1703.9. The identification of the mono-functional H. influenzae Kdo transferase, which is encoded by a KdtA homologue that displays 50% identity to its E. coli counterpart, should facilitate the mechanistic dissection of more complex multi-functional Kdo transferases, like those of E. coli and Chlamydia trachomatis. << Less

  J. Biol. Chem. 272:16555-16563(1997) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Comparative analyses of secondary gene products of 3-deoxy-D-manno-oct-2-ulosonic acid transferases from Chlamydiaceae in Escherichia coli K-12.

  Brabetz W., Lindner B., Brade H.

  The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes ... >> More

  The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and specific monoclonal antibodies. The data show that chlamydial Kdo transferases can replace in E. coli K-12 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that WaaA from C. psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure alpha-Kdo-(2-->8)-[alpha-Kdo-(2-->4)]-alpha-Kdo-(2-->4)-alpha-Kdo. << Less

  Eur. J. Biochem. 267:5458-5465(2000) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.

• A novel 3-deoxy-D-manno-octulosonic acid transferase from Chlamydia trachomatis required for expression of the genus-specific epitope.

  Belunis C.J., Mdluli K.E., Raetz C.R.H., Nano F.E.

  DNA cloned from Chlamydia trachomatis is able to direct the formation of the genus-specific lipopolysaccharide epitope of chlamydiae in enteric Gram-negative bacteria. We now demonstrate that a single C. trachomatis gene (gseA) is sufficient to impart this trait to Escherichia coli. The deduced am ... >> More

  DNA cloned from Chlamydia trachomatis is able to direct the formation of the genus-specific lipopolysaccharide epitope of chlamydiae in enteric Gram-negative bacteria. We now demonstrate that a single C. trachomatis gene (gseA) is sufficient to impart this trait to Escherichia coli. The deduced amino acid sequence of gseA shows 23% identity (66% similarity) to kdtA, an E. coli gene that codes for a bifunctional enzyme catalyzing the addition of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A precursors (Clementz, T., and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 9687-9696). Extracts of E. coli expressing gseA transfer at least one additional Kdo unit from CMP-Kdo to precursors already bearing the two Kdo residues attached by the kdtA gene product. Introduction of gseA into an E. coli mutant with a thermolabile kdtA gene product endows cell extracts with the ability to transfer not only the third but also the first two Kdos to lipid A precursors, demonstrating that the C. trachomatis enzyme is at least trifunctional. Given the similarities of these two Kdo transferases and the essentiality of Kdo in Gram-negative bacteria, lipopolysaccharide biosynthesis may be a target for development of novel drugs effective against chlamydiae. << Less

  J. Biol. Chem. 267:18702-18707(1992) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Bordetella pertussis waaA encodes a monofunctional 2-keto-3-deoxy-D-manno-octulosonic acid transferase that can complement an Escherichia coli waaA mutation.

  Isobe T., White K.A., Allen A.G., Peacock M., Raetz C.R., Maskell D.J.

  Bordetella pertussis lipopolysaccharide (LPS) contains a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue, whereas LPS from Escherichia coli contains at least two. Here we report that B. pertussis waaA encodes an enzyme capable of transferring only a single Kdo during the biosynthesis ... >> More

  Bordetella pertussis lipopolysaccharide (LPS) contains a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue, whereas LPS from Escherichia coli contains at least two. Here we report that B. pertussis waaA encodes an enzyme capable of transferring only a single Kdo during the biosynthesis of LPS and that this activity is sufficient to complement an E. coli waaA mutation. << Less

  J. Bacteriol. 181:2648-2651(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural and mechanistic analysis of the membrane-embedded glycosyltransferase WaaA required for lipopolysaccharide synthesis.

  Schmidt H., Hansen G., Singh S., Hanuszkiewicz A., Lindner B., Fukase K., Woodard R.W., Holst O., Hilgenfeld R., Mamat U., Mesters J.R.

  WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present c ... >> More

  WaaA is a key enzyme in the biosynthesis of LPS, a critical component of the outer envelope of Gram-negative bacteria. Embedded in the cytoplasmic face of the inner membrane, WaaA catalyzes the transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) to the lipid A precursor of LPS. Here we present crystal structures of the free and CMP-bound forms of WaaA from Aquifex aeolicus, an ancient Gram-negative hyperthermophile. These structures reveal details of the CMP-binding site and implicate a unique sequence motif (GGS/TX(5)GXNXLE) in Kdo binding. In addition, a cluster of highly conserved amino acid residues was identified which represents the potential membrane-attachment and acceptor-substrate binding site of WaaA. A series of site-directed mutagenesis experiments revealed critical roles for glycine 30 and glutamate 31 in Kdo transfer. Our results provide the structural basis of a critical reaction in LPS biosynthesis and allowed the development of a detailed model of the catalytic mechanism of WaaA. << Less

  Proc. Natl. Acad. Sci. U.S.A. 109:6253-6258(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.