Atkinson E M, Palcic M M, Hindsgaul O, Long S R
Department of Biological Sciences, Stanford University, CA 94305.
Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8418-22. doi: 10.1073/pnas.91.18.8418.
Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.
根瘤菌合成N-酰化的β-1,4-N-乙酰葡糖胺脂寡糖，即结瘤因子（Nod因子），在固氮根瘤发育过程中，这些因子作为形态发生信号分子作用于豆科植物根系。Nod因子的生物合成在遗传上依赖于结瘤（nod）基因，包括共同的nod基因nodABC。我们利用苜蓿根瘤菌NodH磺基转移酶制备了35S标记的寡糖，用作Nod酶活性的代谢示踪剂。这种方法为追踪壳寡糖修饰提供了一种通用方法。我们发现，N-乙酰壳四糖（壳四糖）单硫酸盐在nodAB依赖下转化为疏水性化合物，通过色谱和化学测试，这些化合物等同于酰化的Nod因子。将标记的中间体与含有NodA或NodB的大肠杆菌进行连续孵育，结果表明在Nod因子生物合成过程中，NodB在NodA之前发挥作用。酰化活性对寡糖链长度敏感，壳四糖作为底物比壳二糖或壳三糖更好。我们通过酶促合成构建了一种假定的Nod因子中间体GlcN-β1,4-(GlcNAc)3，并通过NodH介导的硫酸化对其进行标记，以创建一种特异性代谢探针。这种寡糖的酰化仅需要NodA。这些结果证实了之前关于NodB是一种N-脱乙酰酶的报道，并表明NodA是一种N-酰基转移酶。
