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Taxonomic group: bacteria / Proteobacteria (Phylum: Proteobacteria)
Associated disease: infection due to Escherichia coli [ICD11: XN6P4

]

NCBI PubMed ID: 9446588
Publication DOI: 10.1074/jbc.273.5.2799
Journal NLM ID: 2985121R
Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: raetz

biochem.duke.edu
Institutions: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina

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 inner Kdo residue of Kdo2-lipid IVA as the site of mannosylation. The analog, Kdo-lipid IVA, functions as an acceptor, but is mannosylated at less than 1% the rate of Kdo2-lipid IVA. The purified enzyme displays no activity with ADP-glucose, GDP-mannose, UDP-glucose, or UDP-galactose. Mannosylation of Kdo2-lipid IVA catalyzed by RfaC proceeds in high yield and may be useful for the synthesis of lipopolysaccharide analogs. Pure RfaC can also be used together with Kdo2-[4'-32P]lipid IVA to assay for the physiological donor (presumably ADP-L-glycero-D-manno-heptose) in a crude, low molecular weight fraction isolated from wild type cells.

Lipopolysaccharide, biosynthesis, synthesis, LPS, property, Escherichia, Escherichia coli, heptosyltransferase, enzymatic, purification, enzymatic synthesis

Structure type: oligomer
Location inside paper: Fig. 2
Contained glycoepitopes: IEDB_130650,IEDB_130659,IEDB_130701,IEDB_135394,IEDB_135515,IEDB_141807,IEDB_144983,IEDB_150908,IEDB_151531,IEDB_152206,IEDB_983930,SB_44,SB_67,SB_72

Biosynthesis and genetic data: genetic data,biosynthes data
Synthetic data: biosynthesis

Related record ID(s): 13, 852, 875, 924, 6252, 6306, 6431, 7184, 8403, 10584, 10590, 23706, 24940
NCBI Taxonomy refs (TaxIDs): 83333
Show glycosyltransferases

There is only one chemically distinct structure:

CCCCCCCCC[C@@H](O)CC(=O)N[C@H]1[C@@H](OP(=O)(O)O)O[C@H](CO[C@@H]2O[C@H](CO[C@]3(C(=O)O)C[C@@H](O[C@]4(C(=O)O)C[C@@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O4)[C@@H](O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@@H]([C@H](O)CO)O3)[C@@H](OP(=O)(O)O)[C@H](OC(=O)C[C@H](O)CCCCCCCCC)[C@H]2NC(=O)C[C@H](O)CCCCCCCCC)[C@@H](O)[C@@H]1OC(=O)C[C@H](O)CCCCCCCCC

SVG MW SMILES 3D mol Ionize