螢光影像解析,順式-十一異戊二烯焦磷酸合成酶和細胞膜的交互作用

Abstract

十一異戊二烯焦磷酸合成酶 (Undecaprenyl Pyrophosphate Synthase) 催化八個異戊二烯焦磷酸(isopentenyl pyrophosphate，IPP)和法呢基焦磷酸 (farnesyl pyrophosphate，FPP)反應，生成帶有55個碳元素的十一異戊二烯焦磷酸 (Undecaprenyl Pyrophosphate，UPP)，此產物可以運送脂質到細菌細胞膜上，為細菌細胞壁的主要成分-肽聚糖的前驅物。十一異戊二烯焦磷酸合成酶是水溶性蛋白質，可以在大腸桿菌裡大量表現及純化，而產物十一異戊二烯焦磷酸卻是會嵌入細菌細胞膜。目前尚未得知十一異戊二烯焦磷酸合成酶是如何將十一異戊二烯焦磷酸運送到細菌細胞膜上。為了探討十一異戊二烯焦磷酸合成酶如何將十一異戊二烯焦磷酸運送到細菌細胞膜上的機制，我們使用分子影像相關技術來觀察在不同細胞膜，及相關的受質存在與不存在時，十一異戊二烯焦磷酸合成酶的動態情形。從實驗結果中發現在有無受質的情形下，十一異戊二烯焦磷酸合成酶都可以吸附在由1,2-十四酰基磷脂酰乙醇胺(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine，DMPE)和1,2-十四酰基磷脂酰甘油(1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]，DMPG)以1：3的比例混成的細胞膜上，此細胞膜乃模擬大腸桿菌細胞膜，膜表面帶有負電荷，且可和極性分子產生氫鍵，推測細胞膜表面的電荷是影響十一異戊二烯焦磷酸合成酶可以吸附在膜上的主要因素。另一方面，在合成十一異戊二烯焦磷酸時，十一異戊二烯焦磷酸合成酶可以吸附在由1,2-二棕櫚半合成甘油卵磷脂(1,2-dipalmitoyl-sn-glycero-3-phosphocholine，DPPC )所做成的細胞膜上，此細胞膜表面的淨電荷為零，不會和極性分子產生氫鍵。一旦十一異戊二烯焦磷酸的合成過程被阻止，例如加入法呢基硫代焦磷酸(farnesyl s-thiolodiphosphate，FsPP，此化合物為法呢基焦磷酸的相似物，用來抑制十一異戊二烯焦磷酸的生成)，去除鎂離子，十一異戊二烯焦磷酸合成酶則不能吸附在1,2-二棕櫚半合成甘油卵磷脂細胞膜上。若將十一異戊二烯焦磷酸合成酶上第26號胺基酸-天門冬胺酸(Aspartic acid)突變成丙胺酸(Alanine)，會有部分十一異戊二烯焦磷酸合成酶吸附在1,2-二棕櫚半合成甘油卵磷脂細胞膜上，可見在進行十一異戊二烯焦磷酸合成時，十一異戊二烯焦磷酸合成酶會逐漸吸附到膜上。然而，若將十一異戊二烯焦磷酸合成酶上第83號胺基酸-絲胺酸(Serine)突變成5個丙胺酸[(Ala)5]，這種突變可使十一異戊二烯焦磷酸合成酶維持開啟的狀態，其活性降低介於一千倍到一萬倍，在有無受質存在的情形下，此種突變的十一異戊二烯焦磷酸合成酶可以吸附在1,2-二棕櫚半合成甘油卵磷脂細胞膜上。除此之外，在只有異戊二烯焦磷酸存在的情形下，十一異戊二烯焦磷酸合成酶就可以附著在1,2-二棕櫚半合成甘油卵磷脂細胞膜上。若是去除鎂離子、以及十一異戊二烯焦磷酸合成酶上第26號胺基酸-天門冬胺酸突變成丙胺酸，十一異戊二烯焦磷酸合成酶就不會附著在1,2-二棕櫚半合成甘油卵磷脂細胞膜上。異戊二烯焦磷酸對十一異戊二烯焦磷酸合成酶能不能附著在細胞膜上似乎扮演重要角色。因此，細胞膜表面上的電荷，由不同受質而引起十一異戊二烯焦磷酸合成酶的構形改變，以及異戊二烯焦磷酸，都會影響十一異戊二烯焦磷酸合成酶是否會吸附在細菌細胞膜上。

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes eight consecutive condensation reactions of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C55-Undecaprenyl pyrophosphate (UPP), as a lipid carrier to mediate the synthesis of bacterial cell wall peptidoglycans. UPPs is highly soluble when overexpressed in Escherichia coli, but its product, UPP, is membrane bound. It is unknown how the protein transfers its product to bacterial cell membrane. In order to study the mechanism in which how UPPs adhered to bacterial cell membrane, we employed fluorescent imaging technology to observe the dynamics of UPPs in different cell membranes in the absence and presence of different ligands. From the results, with or without substrates, UPPs always adhered to the membrane mixed with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and 1, 2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) in the ratio of 1:3, which mimicks E. coli. membrane with both negative charges and hydrogen bond-forming groups in it. The net charge in the membrane determines the adhesion of UPPs. On the other hand, during the synthesis of UPP, UPPs adhered to 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane, which had neither net charge nor the polar group which can form hydrogen bonds in it. When the synthesis of UPP was blocked, such as the addition of farnesyl s-thiolodiphosphate (FsPP), which was FPP-analog to inhibit UPPs to synthesize UPP, the elimination of Mg2+, UPPs did not adhere to DPPC membrane. Mutant D26A-UPPs partially adhered to the DPPC membrane in the presence of FPP and IPP, indicating that during the synthesis of UPP, UPPs gradually moves from the defect regions to membrane. However, mutant S83(Ala)5-UPPs, which fixed UPPs in the open form, adhered to DPPC membrane in the absence and presence of the substrates, even though its activity is 103~104 fold lower compared to the wild-type UPPs. In addition, in the presence of IPP, UPPs adhered to DPPC membrane. The elimination of Mg2+ and mutant form of D26A caused UPPs not to adhere to membrane. It seems that IPP binding plays an important role on the adhesion of UPPs to the membrane. Therefore, the net charge of the membrane, the conformational change of UPPs during the synthesis of UPP, and IPP binding determine whether UPPs can adhere to the bacterial cell membrane.