本文關(guān)鍵詞：脂肪酶SMG1小分子抑制劑的篩選及其激活機(jī)制的研究　出處：《華南理工大學(xué)》2016年博士論文　論文類型：學(xué)位論文

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【摘要】：馬拉色球菌CBS 7966來源的脂肪酶SMG1是一個(gè)偏甘油酯脂肪酶。大量研究表明,馬拉色球菌對(duì)頭屑和脂溢性皮炎(D/SD)的致病性與脂肪酶的活性有關(guān),主要是通過脂肪酶介導(dǎo)皮脂的水解,進(jìn)而產(chǎn)生刺激性的游離脂肪酸發(fā)生。因此脂肪酶SMG1是對(duì)抗頭屑和脂溢性皮炎的一個(gè)潛在靶標(biāo)。同時(shí),脂肪酶SMG1還在生物催化中有廣泛的應(yīng)用,包括高純度甘油二酯的合成、磷酯的改造以及烯烴類的環(huán)氧化等,因此有重要有工業(yè)價(jià)值。SMG1的晶體結(jié)構(gòu)顯示,該脂肪酶的催化口袋被一段呈loop結(jié)構(gòu)的“蓋子”區(qū)域覆蓋,因此為閉合結(jié)構(gòu)。SMG1在催化過程中必然經(jīng)歷一定的構(gòu)象變化,使得催化口袋開放,與底物分子結(jié)合。這一構(gòu)象變化過程稱為脂肪酶SMG1的激活機(jī)制。對(duì)SMG1激活機(jī)制的研究,不僅可以從理論角度闡述其結(jié)構(gòu)的柔性對(duì)于催化功能的影響,同時(shí)還可以為理性設(shè)計(jì)、進(jìn)一步改造并提高SMG1的催化活性提供指導(dǎo),以擴(kuò)展SMG1在生物催化領(lǐng)域中的應(yīng)用。本論文以脂肪酶SMG1為研究對(duì)象,旨在于發(fā)現(xiàn)靶向SMG1的抑制劑,為抗頭屑藥物的開發(fā)提供基礎(chǔ);并從分子水平上闡述脂肪酶SMG1在催化過程中的激活機(jī)制,為其催化活性改造提供理論依據(jù)。具體研究內(nèi)容如下:1.脂肪酶SMG1小分子抑制劑的發(fā)現(xiàn)本論文首次以脂肪酶SMG1作為抗頭屑靶標(biāo),展開小分子抑制劑的篩選。篩選的策略為計(jì)算機(jī)輔助的虛擬篩選與基于脂肪酶活性的體外高通量篩選相結(jié)合。在進(jìn)行虛擬篩選前,我們首先采用同源模建的方法搭建了脂肪酶SMG1的蓋子打開的開放結(jié)構(gòu),用于虛擬篩選的對(duì)接。虛擬篩選采用的化合物庫來自Specs公司,其容量為17萬。經(jīng)過篩選和驗(yàn)證,我們獲得了靶向脂肪酶SMG1的首個(gè)先導(dǎo)化合物,它針對(duì)人工底物(對(duì)硝基苯酚乙酯)的IC50為20μM,而針對(duì)天然底物(單油酸甘油酯)的IC50為0.19μM。通過對(duì)該先導(dǎo)化合物類似物的活性評(píng)價(jià)及對(duì)接,我們解析了該先導(dǎo)化合物的結(jié)構(gòu)-活性關(guān)系,并提供了它與SMG1的結(jié)合模式信息。本研究將為后續(xù)以SMG1為靶標(biāo)的更高活性的抑制劑的理性開發(fā)提供一個(gè)起點(diǎn)。2.脂肪酶SMG1的激活機(jī)制研究本論文中關(guān)于脂肪酶SMG1激活機(jī)制的研究分為兩部分。(1)門控機(jī)制。激活機(jī)制的研究策略主要為,通過獲得脂肪酶SMG1催化口袋的開放結(jié)構(gòu),并與閉合結(jié)構(gòu)相對(duì)比,以得出激活過程的構(gòu)象變化。對(duì)SMG1野生型閉合晶體結(jié)構(gòu)的分析可以看出,278和102位殘基連接形成“橋狀”結(jié)構(gòu),將催化口袋從中間截?cái)?并覆蓋了催化三聯(lián)體的絲氨酸。該“橋狀”結(jié)構(gòu)有一定的動(dòng)態(tài)。據(jù)此我們?cè)O(shè)計(jì)了278位的突變體,以較小的殘基代替野生型中較大的苯丙氨酸,來模擬278位殘基向102位殘基的遠(yuǎn)離,期望以此迫使催化口袋變?yōu)殚_放。我們獲得了F278N和F278D的突變體晶體結(jié)構(gòu)。通過結(jié)構(gòu)分析和分子對(duì)接發(fā)現(xiàn),F278N與甘油的復(fù)合晶體結(jié)構(gòu)代表脂肪酶SMG1催化口袋開放的構(gòu)象。通過野生型SMG1的分子動(dòng)力學(xué)模擬以及PMSF抑制實(shí)驗(yàn)的驗(yàn)證得出,脂肪酶SMG1的F278和N102兩個(gè)殘基作為門控開關(guān),控制著SMG1催化口袋的打開和關(guān)閉。門控機(jī)制的闡明為SMG1的理性設(shè)計(jì)提供了依據(jù)。據(jù)此,僅通過單點(diǎn)突變(F278N或F278T),我們?cè)O(shè)計(jì)出了高于野生型SMG1活性6倍的突變體。(2)蓋子區(qū)域的動(dòng)態(tài)在所有的SMG1的晶體結(jié)構(gòu)中,包括閉合的野生型結(jié)構(gòu)和開放的突變體復(fù)合結(jié)構(gòu),均沒有觀察到蓋子結(jié)構(gòu)的構(gòu)象變化,并且蓋子區(qū)域的B-因子(B-factor)相對(duì)于其它區(qū)域較低,因此蓋子結(jié)構(gòu)起初被認(rèn)為在SMG1激活過程保持不變。接下來的研究著重進(jìn)一步驗(yàn)證,除了門控機(jī)制以外,SMG1在激活過程中蓋子區(qū)域到底會(huì)不會(huì)打開。我們采用引入二硫鍵的策略將SMG1的蓋子結(jié)構(gòu)固定于主體骨架中,并通過氧化和還原處理來控制蓋子區(qū)域的固定與否。通過對(duì)蓋子固定和非固定狀態(tài)下的脂肪酶活性測試,我們驗(yàn)證了脂肪酶SMG1的激活過程中,除了102和278兩個(gè)殘基的門控機(jī)制以外,還需要蓋子結(jié)構(gòu)的打開。該研究提示了利用晶體學(xué)研究酶激活過程的缺陷。盡管激活狀態(tài)的晶體結(jié)構(gòu)能夠給出非常豐富的信息,但它畢竟是靜態(tài)的結(jié)構(gòu),并不能反映酶從非活化狀態(tài)到激活狀態(tài)的變化過程。尤其當(dāng)酶激活狀態(tài)的結(jié)構(gòu)能夠局部地恢復(fù)到非活化狀態(tài)時(shí),單獨(dú)依賴晶體學(xué)的研究方法將會(huì)產(chǎn)生嚴(yán)重的后果。本研究中二硫鍵的引入是通過SMG1-L106C/V233C的雙突變體蛋白獲得。我們發(fā)現(xiàn)該突變體與含發(fā)色基團(tuán)的底物相結(jié)合時(shí),可作為一個(gè)定量的巰基氧化還原電勢探針,且在生物催化的級(jí)聯(lián)反應(yīng)中的潛在應(yīng)用。
[Abstract]:Malassezs lipase SMG1 aureus CBS 7966 source is a partial glyceride lipase. Many studies showed that Malassezia aureus against dandruff and seborrheic dermatitis (D/SD) related to the pathogenicity and lipase activity, mainly by lipase mediated hydrolysis of sebum, and free fatty acids stimulate the occurrence of therefore. Lipase SMG1 is a potential target against dandruff and seborrheic dermatitis. At the same time, has been widely used in biological catalysis of lipase SMG1, including synthesis of high purity diacylglycerol, transformation of phosphate esters and olefin epoxidation, so it is important that the crystal structure of industrial value.SMG1, the lipase a catalytic pocket is loop structure of the "lid" coverage, so for the closed structure of.SMG1 must experience some conformational changes in the catalytic process, the catalytic pocket opening, and substrate molecular junctions . this conformational change process called activation mechanism of lipase SMG1. Research on activation mechanism of SMG1, can not only expounds the effects of structural flexibility on the catalytic function from a theoretical point of view, but also for rational design, further reform and improve the catalytic activity of SMG1 to provide guidance to application of extended SMG1 in biocatalysis in the field. This paper using lipase SMG1 as the research object, aims to find targeted inhibitors of SMG1, provide a basis for the development of anti dandruff agents; and from the molecular level on the mechanism of activation in the catalytic process of lipase SMG1, provide a theoretical basis for the catalytic activity of the transformation. The specific contents are as follows: 1. lipase SMG1 inhibitors for the first time with lipase SMG1 as anti dandruff target, start screening of small molecule inhibitors. The screening strategy for virtual screening and computer aided In vitro high-throughput screening of lipase activity in combination. In virtual screening, we use homology model to build an open lid structure of lipase SMG1 open, for docking. Virtual screening using virtual screening of compound libraries from Specs company, its capacity is 170 thousand. After screening and verification, we get targeting the first precursor compound lipase SMG1, the artificial substrate (p-nitrophenol acetate) of IC50 was 20 M, while the natural substrates (glycerol monooleate) IC50 0.19 M. through the evaluation of the active compounds and analogs docking, we analyzed the structure the lead compound activity relationship, and provides the information model combining it with SMG1. To provide a starting point for.2. activation of lipase SMG1 in this study for the subsequent development of rational target SMG1 have higher activity of inhibitors The research on the mechanism of lipase SMG1 activation mechanism is divided into two parts. (1) gating mechanism. Research on activation mechanism is the main strategy, through the open structure of SMG1 by lipase pocket, and compared with the closed structure, with the activation process. The conformational change analysis of the wild type SMG1 closed crystal structure it can be seen that the 278 and 102 residues are connected to form a "bridge" structure, the catalytic pocket from the middle cut, and covered the three CIS catalytic serine. The "bridge" structure has certain dynamic. So we designed 278 mutants, with smaller phenylalanine residues instead of large wild type, to simulate 278 residues away from residue 102, hoping to force the catalytic pocket is open. We obtained a mutant crystal structure of F278N and F278D. Through the analysis of the structure and molecular docking, F2 The conformation of 78N and glycerol composite crystal structure represents the lipase SMG1 catalytic pocket opening. By molecular dynamics simulations of wild-type SMG1 and PMSF inhibition experiments verify that the lipase SMG1 F278 and N102 two residues as gating switch, control of SMG1 catalytic pocket opening and closing. To clarify the gating mechanism provides the basis for for the rational design of SMG1. Therefore, only through a single point mutation (F278N or F278T), we designed the SMG1 activity 6 times higher than that of the wild type mutant. (2) the crystal structure of dynamic lid region in all of the SMG1 in the mutant composite structure including wild type structure and the open closed, no the observed conformational changes of the lid structure, and B- factor covers areas (B-factor) relative to other regions is relatively low, so that the lid structure was initially thought the activation process remained unchanged in the next research SMG1. Weight in addition to further verification, gating mechanism, SMG1 in the activation process in the end will not open lid area. We introduced two disulfide bond strategy will cover the structure of SMG1 is fixed on the main frame, and the oxidation and reduction treatment to control the lid area fixed or not. The lipase activity test on the lid and non fixed the fixed condition, we verify the activation process of lipase SMG1, 102 and 278 in addition to the gating mechanism of two residues, also need to open the lid structure. The research suggests that the defects with crystallographic studies of enzyme activation process. Although the crystal structure active are very rich information, but it is after all is the static structure, and does not reflect the enzyme from inactive state to the active state change process. When the structure of enzyme activation can be partially restored to non activated state, Research methods rely solely on Crystallography will have serious consequences. In this study, the introduction of the two disulfide bonds is obtained by double mutant SMG1-L106C/V233C. We found that the mutant containing chromophore substrate combination, can be used as a quantitative thiol oxidation reduction potential of the probe, and potential applications in cascade biological catalysis in.

【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q55

【參考文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 劉璐;馬拉色菌脂肪酶SMG1酶學(xué)性質(zhì)表征及晶體學(xué)研究[D];華南理工大學(xué);2014年

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本文編號(hào)：1411504