UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype 01 is synthesized by the product of the rfbD(KP01) gene
UDP-galactofuranose precursor required for formation of the lipopolysaccharide O antigen of Klebsiella pneumoniae serotype 01 is synthesized by the product of the rfbD(KP01) gene
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DOI：
10.1074/jbc.272.7.4121
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发表时间：
1997-02-14
影响因子：
4.8
通讯作者：
Whitfield, C
中科院分区：
文献类型：
--
作者：
Koplin, R;Brisson, JR;Whitfield, C
肺炎克雷伯菌O 1脂多糖中的O-侧链多糖基于[-->3）-β-D-Galf-（1-->3）-α-D-Galp-（1-->]重复单元的骨架结构;该结构称为D-半乳聚糖I。rfb（O-抗原生物合成）基因簇指导D-半乳聚糖I的合成，由称为rfbA-F-KPO 1的六个基因组成。在本文中，我们表明rfbD（KPO 1）编码UDP-吡喃半乳糖苷酶（NAD（P）H-需要）（EC 5.4.99.9），它形成尿苷5 '-（二磷酸三氢）P'-α-D-呋喃半乳糖酯（UDP-Galf），呋喃半乳糖残基的生物合成前体。推导的rfbD（KPO 1）基因的氨基酸序列与克氏杆菌rfbD（KPO 8）基因的同源性分别为85%和37.5%。pneumoniae血清型O 8和大肠杆菌的glf基因。纯化的RfbD（KPO 1）酶的分子量为45 kDa，通过SDS-聚丙烯酰胺凝胶电泳测定，而凝胶过滤显示的分子量为92 kDa，表明天然蛋白质的二聚体结构。rfbD（KPO 1）基因产物可相互转化尿苷5 '-（二磷酸三氢）P'-α-D-吡喃半乳糖基酯（UDP-Galp）和UDP-Galf：与Glf不同，RfbD（KPO 1）需要NADH或NADPH，不能被NAD或NADP取代。RfbD（KPO 1）用于合成毫克量的UDP-Galf，使该化合物首次以完整形式纯化和充分表征。通过NMR光谱证实了UDP-Galf的结构。
The O-side-chain polysaccharide in the Lipopolysaccharide of Klebsiella pneumoniae O1 is based on a backbone structure of repeat units of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; this structure is termed D-galactan I. The rfb (O-antigen biosynthesis) gene cluster directs the synthesis of D-galactan I and consists of six genes termed rfbA-F-KPO1. In this paper we show that rfbD(KPO1) encodes a UDP-galactopyranose mutase (NAD(P)H-requiring) (EC 5.4.99.9), which forms uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactofuranosyl ester (UDP-Galf), the biosynthetic precursor of galactofuranosyl residues. The deduced amino acid sequence of rfbD(KPO1) shows 85% and 37.5% identity to the rfbD(KPO8) gene of K. pneumoniae serotype O8 and the glf gene of Escherichia coli, respectively. The molecular mass of the purified RfbD(KPO1) enzyme is 45 kDa as determined by SDS-polyacrylamide gel electrophoresis, while gel filtration revealed a molecular mass of 92 kDa, suggesting a dimeric structure for the native protein. The rfbD(KPO1) gene product interconverts uridine 5'-(trihydrogen diphosphate) P'-alpha-D-galactopyranosyl ester (UDP-Galp) and UDP-Galf: Unlike Glf, RfbD(KPO1) showed a requirement for NADH or NADPH, which could not be replaced by NAD or NADP. RfbD(KPO1) was used to synthesize milligram quantities of UDP-Galf, allowing this compound to be purified and fully characterized in an intact form for the first time. The structure of UDP-Galf was proven by NMR spectroscopy.