生物谷：豆蔻酰輔酶A：蛋白質(zhì)N-豆蔻酰轉(zhuǎn)移酶（NMT）負(fù)責(zé)催化將豆蔻酰輔酶A上的豆蔻酸共價(jià)連接到真核生物和病毒的眾多新生蛋白N末端的甘氨酸上,，在多種重要細(xì)胞過(guò)程中發(fā)揮重要的生物學(xué)功能。遺傳和生化研究結(jié)果表明NMT是抗真菌藥物的理想靶標(biāo),。在過(guò)去的十年中多個(gè)實(shí)驗(yàn)室對(duì)兩個(gè)種屬的NMTs進(jìn)行了比較深入的結(jié)構(gòu)和功能研究,，但在獲得的晶體結(jié)構(gòu)中由于NMT的N端區(qū)域的缺失或無(wú)法定位,，其生物學(xué)功能還不清楚。
生化與細(xì)胞所丁建平研究組與美國(guó)杜邦公司Stine Haskell研究中心Steven Gutteridge研究組經(jīng)過(guò)三年多的合作,，運(yùn)用結(jié)構(gòu)生物學(xué)和生物化學(xué)的手段闡明了酵母NMT（ScNMT）的N端區(qū)域的生物學(xué)功能和酶催化反應(yīng)的分子機(jī)制,，為設(shè)計(jì)和研制更加有效的抗真菌藥物提供了有價(jià)值的生物學(xué)信息和結(jié)構(gòu)基礎(chǔ)。上述研究成果得到同行評(píng)審專家和雜志編輯的高度贊賞,，已于2007年5月18日在線發(fā)表于《The Journal of Biological Chemistry》雜志,。
研究者們解析了全長(zhǎng)ScNMT與豆蔻酰輔酶A的二元復(fù)合物的晶體結(jié)構(gòu)，清晰地確定了以往報(bào)道中從未觀察到的ScNMT的N末端區(qū)域的結(jié)構(gòu),，該區(qū)域緊靠酶催化反應(yīng)中心,，參與了豆蔻酰輔酶A和底物的識(shí)別和結(jié)合。研究者們進(jìn)一步構(gòu)建了一系列含有重要氨基酸點(diǎn)突變和截短形式的ScNMT突變體,，運(yùn)用生物化學(xué)的方法測(cè)定了它們的酶催化反應(yīng)的各項(xiàng)動(dòng)力學(xué)參數(shù),。基于結(jié)構(gòu)生物學(xué)和生物化學(xué)的分析結(jié)果,，研究者們揭示了NMT的N端區(qū)域參與底物和輔酶A的識(shí)別與結(jié)合,，但并不影響最終的酶促反應(yīng)活性。同時(shí),，這些研究結(jié)果還澄清了文獻(xiàn)報(bào)道中關(guān)于其它結(jié)構(gòu)元素和氨基酸參與底物識(shí)別和結(jié)合功能的預(yù)測(cè)和假設(shè),。
另外，研究者們還解析了全長(zhǎng)ScNMT與豆蔻酰輔酶A和兩種高活性抑制劑形成的三元復(fù)合物的晶體結(jié)構(gòu),，確定了抑制劑占據(jù)著底物多肽的結(jié)合位點(diǎn),，從而發(fā)揮抑制酶活的功能,。通過(guò)對(duì)抑制劑與酶相互作用的分析,、以及與其它結(jié)構(gòu)的比較，研究者們發(fā)現(xiàn)多肽底物的結(jié)合口袋具有較高的剛性,，但部分氨基酸殘基的側(cè)鏈會(huì)根據(jù)抑制劑的化學(xué)性質(zhì)和結(jié)構(gòu)的不同發(fā)生微小的構(gòu)象調(diào)整,。據(jù)此，研究者們提出了對(duì)現(xiàn)有的抑制劑進(jìn)行化學(xué)修飾和結(jié)構(gòu)改造的思路,，以增加它們與酶的相互作用和特異性,。這些結(jié)果為研究和開(kāi)發(fā)具有更高結(jié)合能力和抑制活性的抗真菌藥物提供了很好的結(jié)構(gòu)基礎(chǔ)和理論指導(dǎo)。（引自生化細(xì)胞所）
原始出處:
Papers In Press, published online ahead of print May 18, 2007
J. Biol. Chem, 10.1074/jbc.M702696200
Submitted on March 29, 2007
Revised on May 17, 2007
Accepted on May 18, 2007
Crystal structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoyl-CoA and inhibitors reveal the functional roles of the N-terminal region
Jian Wu, Yong Tao, Meilan Zhang, Michael Howard, Steven Gutteridge, and Jianping Ding
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai 200031
Corresponding Author: [email protected]
Protein N-myristoylation catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) plays an important role in a variety of critical cellular processes and thus is an attractive target for development of antifungal drugs. We report here three crystal structures of Saccharomyces. cerevisiae NMT; as a binary complex with myristoyl-CoA alone and two ternary complexes involving myristoyl-CoA and two different non-peptidic inhibitors. In all three structures, the majority of the N-terminal region, absent in all previously reported structures, forms a well defined motif that is located in the vicinity of the peptide substrate binding site and is involved in the binding of myristoyl-CoA. The Ab loop, which might be involved in substrate recognition, adopts an open conformation; while a loop of the N-terminal region (residues 22-24) that covers the top of the substrate binding site, is in the position occupied by the Ab loop when in the closed conformation. Structural comparisons with other NMTs, together with mutagenesis data suggest that the N-terminal region of NMT plays an important role in the binding of both myristoyl-CoA and peptide substrate, but not in subsequent steps of the catalytic mechanism. The two inhibitors are found occupying the peptide substrate binding site and interact with the protein through primarily hydrophobic contacts. Analyses of the inhibitor-enzyme interactions provide valuable information for further improvement of antifungal inhibitors targeting NMT.
