Zayni Sonja, Steiner Kerstin, Pföstl Andreas, Hofinger Andreas, Kosma Paul, Schäffer Christina, Messner Paul
Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1190 Vienna, Austria.
Glycobiology. 2007 Apr;17(4):433-43. doi: 10.1093/glycob/cwl084. Epub 2007 Jan 3.
The glycan chain of the S-layer protein of Geobacillus tepidamans GS5-97(T) consists of disaccharide repeating units composed of L-rhamnose and D-fucose, the latter being a rare constituent of prokaryotic glycoconjugates. Although biosynthesis of nucleotide-activated L-rhamnose is well established, D-fucose biosynthesis is less investigated. The conversion of alpha-D-glucose-1-phosphate into thymidine diphosphate (dTDP)-4-dehydro-6-deoxyglucose by the sequential action of RmlA (glucose-1-phosphate thymidylyltransferase) and RmlB (dTDP-glucose-4,6-dehydratase) is shared between the dTDP-D-fucose and the dTDP-L-rhamnose biosynthesis pathway. This key intermediate is processed by the dTDP-4-dehydro-6-deoxyglucose reductase Fcd to form dTDP-alpha-D-fucose. We identified the fcd gene in G. tepidamans GS5-97(T) by chromosome walking and performed functional characterization of the recombinant 308-amino acid enzyme. The in vitro activity of the enzymatic cascade (RmlB and Fcd) was monitored by high-performance liquid chromatography and the reaction product was confirmed by (1)H and (13)C nuclear magnetic resonance spectroscopy. This is the first characterization of the dTDP-alpha-D-fucopyranose biosynthesis pathway in a Gram-positive organism. fcd was identified as 1 of 20 open reading frames contained in a 17471-bp S-layer glycosylation (slg) gene cluster on the chromosome of G. tepidamans GS5-97(T). The sgtA structural gene is located immediately upstream of the slg gene cluster with an intergenic region of 247 nucleotides. By comparison of the SgtA amino acid sequence with the known glycosylation pattern of the S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a, two out of the proposed three glycosylation sites on SgtA could be identified by electrospray ionization quadrupole-time-of-flight mass spectrometry to be at positions Ser-792 and Thr-583.
嗜热栖热放线菌GS5-97(T)的S层蛋白聚糖链由L-鼠李糖和D-岩藻糖组成的二糖重复单元构成，后者是原核糖缀合物中的稀有成分。尽管核苷酸活化L-鼠李糖的生物合成已得到充分证实，但D-岩藻糖的生物合成研究较少。RmlA（葡萄糖-1-磷酸胸苷酰转移酶）和RmlB（dTDP-葡萄糖-4,6-脱水酶）的顺序作用将α-D-葡萄糖-1-磷酸转化为胸苷二磷酸（dTDP）-4-脱氢-6-脱氧葡萄糖，这在dTDP-D-岩藻糖和dTDP-L-鼠李糖生物合成途径中是相同的。这个关键中间体由dTDP-4-脱氢-6-脱氧葡萄糖还原酶Fcd加工形成dTDP-α-D-岩藻糖。我们通过染色体步移在嗜热栖热放线菌GS5-97(T)中鉴定了fcd基因，并对重组的308个氨基酸的酶进行了功能表征。通过高效液相色谱监测酶促级联反应（RmlB和Fcd）的体外活性，并通过1H和13C核磁共振光谱确认反应产物。这是革兰氏阳性生物中dTDP-α-D-呋喃岩藻糖生物合成途径的首次表征。fcd被鉴定为嗜热栖热放线菌GS5-97(T)染色体上17471 bp的S层糖基化（slg）基因簇中包含的20个开放阅读框之一。sgtA结构基因位于slg基因簇的紧上游，基因间隔区为247个核苷酸。通过将SgtA氨基酸序列与嗜热栖热放线菌NRS 2004/3a的S层蛋白SgsE的已知糖基化模式进行比较，通过电喷雾电离四极杆-飞行时间质谱法可以确定SgtA上三个提议的糖基化位点中的两个位于Ser-792和Thr-583位置。
