Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from Escherichia coli

Abstract

Heptosyltransferase II, encoded by the waaF gene of Escherichia coli, is a glycosyltransferase involved in the synthesis of the inner core region of lipopolysaccharide. The gene was subcloned from plasmid pWSB33 [Brabetz, W., Mu Èller-Loennies, S., Holst, O. & Brade, H. (1997) Eur. J. Biochem. 247, 716±724] into a shuttle vector for the expression in the gram-positive host Corynebacterium glutamicum. The in vitro activity of the enzyme was investigated in comparison to that of heptosyltransferase I (WaaC) using as a source for the sugar nucleotide donor, ADP-l-glycero-d-manno-heptose, a low molecular mass filtrate from a DwaaCF E. coli strain. Synthetic lipid A analogues varying in the acylation or phosphorylation pattern or both were tested as acceptors for the subsequent transfer of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and heptose by successive action of Kdo transferase (WaaA), heptosyltransferase I (WaaC) and heptosyltransferase II (WaaF). The reaction products were characterized after separation by TLC and blotting with monoclonal antibodies specific for the acceptor, the intermediates and the final products.

Lipopolysaccharide Endotoxins

“…Using synthetic substrates and in vitro reactions, it has been confirmed that the configuration of L,D-Hep residues in the core is established by the specificity of the heptosyltransferases, WaaC and WaaF. These enzymes exhibit preference for ADP-L,D-heptose as their substrate, with ADP-D,D-heptose being used at low efficiency (208,209). Interestingly, ADP-Dmannose can also serve as a surrogate substrate for WaaC (210).…”

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12

“…The first three gene products are involved in the biosynthesis of the LPS inner core. waaF encodes ADP-heptose : LPS heptosyltransferase II (Gronow, et al, 2000). The product of the waaL gene, Oantigen ligase, connects the inner core to the O antigen of LPS (Klena et al, 1992).…”

Biosynthesis of a Novel 3-Deoxy-D-manno-oct-2-ulosonic Acid-containing Outer Core Oligosaccharide in the Lipopolysaccharide of Klebsiella pneumoniae

“…Purification of CMP-Kdo Synthetase (KdsB)-Plasmid pJKB72 (23) was transformed into E. coli LE392 for overexpression of KdsB. The purification procedure was a modification of that described elsewhere (23). The strain was grown for 18 h at 37°C in LB medium supplemented with kanamycin.…”

“…Kdo Transferase Activity of WabI-The enzymatic activity of WabI was assayed in a standard reaction adapted from the procedure of Gronow et al (23). The 0.05-ml reaction mixture comprised 50 mM HEPES (pH 7.5) containing 10 mM MgCl 2 , 2 mM Kdo, 5 mM CTP, 0.3 mg of CWG600 (wabI) mutant LPS acceptor, 0.007 mg of KdsB, and either 0.2 mg (protein) of the soluble cell-free lysate from the BL21(DE3)(pET28) control or BL21(DE3)(pWQ40(His 6 -WabI ϩ )) or 0.004 mg of purified His 6 -WabI.…”