Sulfolipid Head Group Biosynthesis in Photosynthetic Organisms

[807943] benning含有丰富的非磷亚脂类磺基喹啉二酰基甘油，与细菌和植物的光合膜有关。独特的头部基团由葡萄糖的6-磺酸盐衍生物组成，称为磺喹诺糖。脂肪酸的生物合成有三个主要途径：碳水化合物代谢、硫同化和脂肪酸生物合成。有充分的证据表明，巯基喹啉的最终组装是由巯基喹啉部分从udp -磺基喹啉转移到二酰基甘油的sn-3位置。然而，对于前体udp -磺基喹诺糖的生物合成和引入磺酸基的机制仍然知之甚少。本项目的目的是阐明udp -磺基喹诺糖的生物合成，从而确定巯基脂的生物合成途径，并可能发现一种对生物系统中磺酸盐合成至关重要的新的生化反应。一种方法是鉴定光合细菌的sqdB亚脂基因或植物的同源SQD1基因所编码的蛋白，并阐明其催化的代谢反应。由于这些蛋白的氨基酸序列与糖核苷酸修饰酶的氨基酸序列相似，因此被认为参与了由udp -葡萄糖合成udp -磺基藜糖的生物合成。重组SQDB或SQD1蛋白在体外将udp -葡萄糖转化为新的化合物。该化合物可能是形成udp -磺基喹诺糖的硫受体，其结构解析有望为体内SQDB或SQD1蛋白催化的反应提供线索。第二种方法旨在鉴定可能用于udp -磺基喹诺糖生物合成的硫供体，从而确定一般硫代谢和硫脂生物合成之间的分支点。为此，我们将通过靶向基因破坏构建4个缺乏不同硫酸盐同化和还原步骤的蓝细菌Synechocystis sp. PCC6803突变体，并对其合成硫酸脂的能力进行测试。巯基脂是细菌和植物光合膜的组成成分。巯基脂的头基团是独特的，由葡萄糖的磺酸衍生物称为磺喹诺糖。这个项目的目的是阐明导致这种自然发生的磺酸的生物合成的关键生化反应。编码酶的基因被认为可以催化头基团的生物合成，这些基因可以从细菌和植物中获得。这些基因被用来在大肠杆菌中产生重组蛋白，可以对其进行详细的研究，以阐明反应机制。为了解决磺胺中硫的起源问题，已知的编码蛋白质的基因参与了蓝藻中一般硫代谢的不同反应，将被灭活。由此产生的突变体将被测试其对亚脂生物合成的能力。对巯基生物合成的新认识不仅解决了巯基生物合成的途径，而且可能为通过基因工程生产磺酸提供线索。在不同的实验室试验中已证明，亚脂具有抗病毒和抗肿瘤活性。因此，在不久的将来，以廉价的方式大量生产该化合物可能是可取的。

9807943Benning The abundant non-phosphorous sulfolipid sulfoquinovosyl diacylglycerol is specifically associated with photosynthetic membranes of bacteria and plants. The unique head group consists of a 6-sulfonate derivative of glucose called sulfoquinovose. Three major pathways contribute to the biosynthesis of sulfolipid: carbohydrate metabolism, sulfur assimilation, and fatty acid biosynthesis. There is good evidence for the final assembly of the sulfolipid by the transfer of the sulfoquinovosyl moiety from UDP-sulfoquinovose to the sn-3 position of diacylglycerol. However, very little is still known about the biosynthesis of the precursor UDP-sulfoquinovose and the mechanism for the introduction of the sulfonate group. It is the objective of this project, to elucidate the biosynthesis of UDP-sulfoquinovose, thereby determining the pathway of sulfolipid biosynthesis and possibly discovering a novel biochemical reaction crucial to the synthesis of sulfonates in biological systems. One approach is to characterize the proteins encoded by the sqdB sulfolipid genes of photosynthetic bacteria or by the orthologous SQD1 plant gene, and to elucidate the metabolic reaction catalyzed. These proteins are proposed to be involved in the biosynthesis of UDP-sulfoquinovose from UDP-glucose, because their amino acid sequence is similar to those of sugar nucleotide modifying enzymes. Recombinant SQDB or SQD1 proteins convert UDP-glucose to a new compound in vitro. The structural elucidation of this compound, presumably the sulfur acceptor for the formation of UDP-sulfoquinovose, is expected to provide clues to the reaction catalyzed by the SQDB or SQD1 proteins in vivo. A second approach is aimed at the identification of the possible sulfur donor for UDP-sulfoquinovose biosynthesis and, thus, the branch point between general sulfur metabolism and sulfolipid biosynthesis. For this purpose, four mutants of the cyanobacterium Synechocystis sp. PCC6803 deficient in different steps of sulfate assimilation and reduction will be constructed by targeted gene disruption and will be tested for their capability to synthesize sulfolipid. Sulfolipid is a constituent of photosynthetic membranes of bacteria and plants. The head group of the sulfolipid is unique, consisting of a sulfonic acid derivative of glucose called sulfoquinovose. It is the objective of this project to elucidate the crucial biochemical reaction(s) leading to the biosynthesis of this naturally occurring sulfonic acid. The genes encoding the enzyme thought to catalyze the biosynthesis of the head group are available from bacteria and plants. These genes are used to produce recombinant proteins in E. coli that can be studied in detail to elucidate the reaction mechanism. To address the question for the origin of the sulfur in sulfolipid, genes known to encode proteins involved in different reactions of the general sulfur metabolism in cyanobacteria will be inactivated. The resulting mutants will be tested for their competence for sulfolipid biosynthesis. The newly acquired knowledge about the biosynthesis of the sulfolipid head group will not only solve the pathway of sulfolipid biosynthesis, but may also provide clues towards the production of sulfonic acids by genetic engineering. It has been demonstrated in different laboratory tests that sulfolipid has anti-viral and anti-tumor activities. Thus, it may become desirable to produce large quantities of this compound in an inexpensive way in the near future.