Functional Assignment of an Enzyme that Catalyzes the Synthesis of an Archaea-Type Ether Lipid in Bacteria
Functional Assignment of an Enzyme that Catalyzes the Synthesis of an Archaea-Type Ether Lipid in Bacteria
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DOI：
10.1002/anie.201101832
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发表时间：
2011-01-01
影响因子：
16.6
通讯作者：
Babinger, Patrick
中科院分区：
文献类型：
--
作者：
Guldan, Harald;Matysik, Frank-Michael;Babinger, Patrick
宇宙生命树将所有生物体分为真核生物、细菌和古生菌三个系统发育域。[1]这些结构域之间的主要区别是形成细胞膜的脂质的化学组成。[2-5]来自细菌和真核生物的磷脂由sn-甘油-3-磷酸（G3 P）核心组成，脂肪酸通过酯键与之结合，而来自古细菌的磷脂由sn-甘油-1-磷酸（G1 P）和两个通过醚键连接的类异戊二烯链组成（图1）。这种差异表明，古生菌在进化过程中的出现与甘油-1-磷酸脱氢酶（G1 PDH）和香叶基香叶基甘油磷酸合酶（GGGPS）的出现有关。[6-9]这些连续作用的酶催化导致基于G1 P的醚脂质的前两个步骤，即磷酸二羟丙酮（DHAP）还原为G1 P及其与活化的类异戊二烯香叶基香叶基焦磷酸（GGPP; 20个碳原子）的缩合，产生香叶基香叶基甘油基磷酸（GGGP）。GGGP然后逐步转化为基于G1 P的醚脂质，如图1所示。由于G1 P和GGGP被认为是古细菌的典型特征，因此在某些细菌物种中发现与G1 PDH和GGGPS具有显著序列相似性的蛋白质是出乎意料的。[6，10]我们对鉴定这些蛋白质的功能感兴趣，并且最近表明，来自革兰氏阳性细菌枯草芽孢杆菌的AraM酶与古细菌G1 PDH同源，催化DHAP的NADH+依赖性还原为G1 P。[11]我们现在已经破译了细菌PcrB家族的功能，其成员是古细菌GGGPS的同源物。我们的方法是基于酶的能力，以转换放射性标记的G1 P与第二个，迄今未知的聚异戊二烯基底物提供的B。枯草杆菌细胞所形成的产物的表征表明，PcrB在体内催化G1 P与焦磷酸七异戊二烯酯（HepPP; 35个碳原子）缩合成磷酸七异戊二烯甘油酯（HepGP）。HepGP，这是第一个古菌型G1 P为基础的醚脂质被确定在细菌的系统发育域，被发现随后去磷酸化和乙酰化。此外，我们表明，不同的底物特异性的古细菌GGGPS和细菌PcrB，结合polyprenyl部分含有20和35个碳原子，分别是由一个单一的氨基酸差异底部的活性位点。
The universal tree of life divides all organisms into the three phylogenetic domains eukaryota, bacteria, and archaea.[1] A main difference between these domains is the chemical composition of the lipids forming cellular membranes.[2–5] Phospholipids from bacteria and eukaryota are composed of a sn-glycerol-3-phosphate (G3P) core to which fatty acids are bound via ester linkages, while phospholipids from archaea consist of a sn-glycerol-1-phosphate (G1P) with two isoprenoid chains attached by ether bonds (Figure 1). This difference suggests that the emergence of the archaea during evolution was linked to the advent of glycerol-1-phosphate dehydrogenase (G1PDH) and geranylgeranylglyceryl phosphate synthase (GGGPS).[6–9] These consecutively acting enzymes catalyze the first two steps leading to G1P-based ether lipids, which are the reduction of dihydroxyacetone phosphate (DHAP) to G1P and its condensation with the activated isoprenoid geranylgeranyl pyrophosphate (GGPP; 20 carbon atoms), giving rise to geranylgeranylglyceryl phosphate (GGGP). GGGP is then stepwise converted into G1P-based ether lipids, such as the one shown in Figure 1. As G1P and GGGP are considered to be typical of archaea, the discovery of proteins with significant sequence similarities to G1PDH and GGGPS within certain species of the bacteria was unexpected.[6, 10] We are interested in identifying the function of these proteins, and recently showed that the AraM enzyme from the gram-positive bacterium Bacillus subtilis, which is homologous to the archaeal G1PDH, catalyzes the NADH+-dependent reduction of DHAP to G1P.[11]We have now deciphered the function of the bacterial PcrB family, whose members are homologues of the archaeal GGGPS. Our approach was based on the ability of the enzyme to convert radioactively labeled G1P with a second, hitherto unknown polyprenyl substrate being provided by B. subtilis cells. The characterization of the formed products demonstrates that PcrB catalyzes in vivo the condensation of G1P with heptaprenyl pyrophosphate (HepPP; 35 carbon atoms) to heptaprenylglyceryl phosphate (HepGP). HepGP, which is the first archaea-type G1P-based ether lipid being identified within the phylogenetic domain of the bacteria, was found to be subsequently dephosphorylated and acetylated. Moreover, we show that the different substrate specificities of the archaeal GGGPS and the bacterial PcrB, which bind polyprenyl moieties containing 20 and 35 carbon atoms, respectively, are caused by a single amino acid difference at the bottom of the active site.