期刊
BIOCHEMISTRY
卷 48, 期 6, 页码 1162-1172出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi802211k
关键词
-
资金
- NIH [R-37-GM-51796, R01 A1057588]
- LIPID MAPS Large Scale Collaborative [GM-069338]
- NSFC [30770114]
- 111 Project [111-2-06]
- NCI [P30-CA-14236]
- NSF
- NC Biotechnology Center
- Duke University
Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. On the basis of electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-beta-D-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin-layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M-H](-) at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. P-31 NMR spectroscopy showed a single phosphorus atom in the phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. H-1 NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a beta-D-GalN unit. Heteronuclear multiple-quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-beta-D-GalN supports the in vitro modification of lipid IVA by membranes of Escherichia coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4-amino-4-deoxy-L-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-beta-D-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据