Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose.
Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose.
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耻垢分枝杆菌中 d-阿拉伯糖的生物合成：d-葡萄糖的特异性标记。
DOI：
10.1006/abbi.2001.2723
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Elbein,AlanD
中科院分区：
文献类型：
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作者：
Klutts,JStacey;Hatanaka,Kenichi;Pan,YT;Elbein,AlanD
d-阿拉伯糖是结核分枝杆菌和其他分枝杆菌物种的细胞壁多糖中的主要糖。参与d-阿拉伯糖的生物合成和活化的反应代表了药物干预的极好的潜在位点，因为在哺乳动物细胞中未发现d-阿拉伯糖，并且细胞壁阿拉伯甘露聚糖和/或阿拉伯半乳聚糖似乎是细胞存活所必需的。由于d-葡萄糖转化为d-阿拉伯糖的途径尚不清楚，我们将耻垢分枝杆菌细胞分别与[1- 14 C]葡萄糖、[3，4 - 14 C]葡萄糖和[6- 14 C]葡萄糖孵育，并比较细胞壁结合的阿拉伯糖的比活性。尽管使用[6- 14 C]葡萄糖时阿拉伯糖的比活性比使用其他标记物时低约25%，但似乎没有选择性损失碳1或碳6，表明阿拉伯糖不是通过戊糖磷酸途径的氧化步骤损失葡萄糖的碳1或通过糖醛酸途径中的碳6形成的。用从核酸级分分离的核糖观察到类似的标记模式。由于这些结果表明戊糖形成的不寻常途径，还用[1- 13 C]葡萄糖、[2- 13 C]葡萄糖和[6- 13 C]葡萄糖进行标记研究，并通过NMR分析检查细胞壁阿拉伯糖。这种方法可以测定阿拉伯糖中每个碳原子的相对13 C含量。标记模式表明，最可能的途径是由转醛醇酶反应产生的果糖6-磷酸的碳1和2与碳4、5和6缩合（即，甘油醛3-磷酸）由果糖-1，6二磷酸醛缩酶形成。M.在各种实验条件下，将耻垢病与核糖5-磷酸、木酮糖5-磷酸和D-阿拉伯糖5-磷酸一起孵育。尽管核糖5-磷酸和木酮糖5-磷酸被转化为其他戊糖和己糖，但在这些反应中的任何一个中都没有检测到阿拉伯糖5-磷酸（或游离阿拉伯糖）。此外，这些酶提取物不将阿拉伯糖5-磷酸转化为任何其他戊糖或己糖。此外，[14 C]葡萄糖6-磷酸和各种核苷三磷酸（ATP、CTP、GTP、TTP和UTP）与来自分枝杆菌细胞的胞质或膜组分一起孵育不会导致形成阿拉伯糖的核苷酸形式，尽管在核苷酸组分中发现了其他放射性糖，包括鼠李糖和半乳糖。此外，在从M分离的核苷酸组分中未发现放射性阿拉伯糖。在[3 H]葡萄糖中生长的耻垢病细胞中，在从300 g细胞中大规模提取糖核苷酸级分时也未检测到阿拉伯糖。从这些研究中得出的逻辑结论是，d-阿拉伯糖可能是由d-核糖通过聚异戊二烯磷酸-核糖的核糖部分的碳2差向异构化形成聚异戊二烯磷酸-阿拉伯糖，然后将其用作形成阿拉伯糖基聚合物的前体而产生的。
d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-14C]glucose, [3,4-14C]glucose, and [6-14C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-14C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-13C]glucose, [2-13C]glucose, and [6-13C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative13C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [14C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [3H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.