Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i.

The Journal of Biological Chemistry
J L Kadrmas, C R Raetz

Abstract

Heptosyltransferase I, encoded by the rfaC(waaC) gene of Escherichia coli, is thought to add L-glycero-D-manno-heptose to the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide core. Lipopolysaccharide isolated from mutants defective in rfaC lack heptose and all other sugars distal to heptose. The putative donor, ADP-L-glycero-D-manno-heptose, has never been fully characterized and is not readily available. In cell extracts, the analog ADP-mannose can serve as an alternative donor for RfaC-catalyzed glycosylation of the acceptor, Kdo2-lipid IVA. Using a T7 promoter construct that overexpresses RfaC approximately 15,000-fold, the enzyme has been purified to near homogeneity. NH2-terminal sequencing confirms that the purified enzyme is the rfaC gene product. The subunit molecular mass is 36 kDa. Enzymatic activity is dependent upon the presence of Triton X-100 and is maximal at pH 7.5. The apparent Km (determined at near saturating concentrations of the second substrate) is 1.5 mM for ADP-mannose and 4.5 microM for Kdo2-lipid IVA. Chemical hydrolysis of the RfaC reaction product at 100 degrees C in the presence of sodium acetate and 1% sodium dodecyl sulfate generates fragments consistent with the inne...Continue Reading

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Citations

Feb 25, 2006·The Journal of Biological Chemistry·Suparna Kanjilal-KolarChristian R H Raetz
Aug 7, 2007·Genome Research·Lise PetersenRasmus Nielsen
Jun 5, 2002·Annual Review of Biochemistry·Christian R H Raetz, Chris Whitfield
Jan 7, 2009·Bioorganic & Medicinal Chemistry·Nicolas DesroySonia Escaich
Jul 29, 2008·Research in Microbiology·Marie-Eve Charbonneau, Michael Mourez
Oct 6, 2000·Annual Review of Microbiology·M Riley, M H Serres
Nov 9, 2016·Frontiers in Microbiology·Bram VivijsChris W Michiels
Jan 16, 2016·PloS One·Sophie J WeissRyan T Gill
Oct 28, 2017·International Journal of Molecular Sciences·Joy M Cote, Erika A Taylor
Apr 12, 2014·The Journal of Biological Chemistry·Gracjana KleinSatish Raina
Nov 26, 2019·Annals of the New York Academy of Sciences·Craig R MacNairEric D Brown
Dec 26, 2001·Journal of Bacteriology·Bernd KneidingerPaul Messner

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