Riekhof Wayne R, Sears Barbara B, Benning Christoph
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Eukaryot Cell. 2005 Feb;4(2):242-52. doi: 10.1128/EC.4.2.242-252.2005.
Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.
开花植物中的脂质代谢已得到深入研究，关于编码主要脂肪酸和膜脂生物合成途径成分的基因的信息非常丰富。我们现在展示了对一种藻类模型中脂肪酸和甘油脂代谢的计算机分析，这得益于莱茵衣藻表达序列标签和基因组序列的近期可得性。基于与具有已确认功能的蛋白质的相似性，预测了参与膜生物发生的蛋白质编码基因，并进行了组织，以重建衣藻中甘油脂合成的主要途径。该分析涵盖了预计编码参与膜脂生物合成合成反应的酶的大多数基因，并比较了衣藻和开花植物中的这些途径。作为生物信息学分析的一个重要结果，我们鉴定并分离了莱茵衣藻BTA1（BTA1Cr）基因，并分析了它编码的双功能蛋白；我们预测该蛋白足以合成甜菜碱脂二酰甘油 - N，N，N - 三甲基高丝氨酸（DGTS），这是衣藻中的一种主要膜成分。BTA1Cr的异源表达导致DGTS在通常缺乏这种脂质的大肠杆菌中积累，并允许对BTA1Cr的酶学性质进行体外分析。相比之下，在球形红细菌中，DGTS的生物合成需要两种单独的蛋白质，BtaARs和BtaBRs。对BTA1Cr两个结构域的活性位点进行定点诱变使我们能够分别研究它们的活性，直接证明它们与细菌直系同源物BtaARs和BtaBRs的功能同源性。
