豆蔻醯輔酶A:蛋白質N-豆蔻醯轉移酶的N端區域的生物學功能和藥物設計

    生物谷：豆蔻醯輔酶A：蛋白質N-豆蔻醯轉移酶（NMT）負責催化將豆蔻醯輔酶A上的豆蔻酸共價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，在多種重要細胞過程中發揮重要的生物學功能。遺傳和生化研究結果表明NMT是抗真菌藥物的理想靶標。在過去的十年中多個實驗室對兩個種屬的NMTs進行了比較深入的結構和功能研究，但在獲得的晶體結構中由於NMT的N端區域的缺失或無法定位，其生物學功能還不清楚。

    生化與細胞所丁建平研究組與美國杜邦公司Stine  Haskell研究中心Steven  Gutteridge研究組經過三年多的合作，運用結構生物學和生物化學的手段闡明了酵母NMT（ScNMT）的N端區域的生物學功能和酶催化反應的分子機制，為設計和研製更加有效的抗真菌藥物提供了有價值的生物學信息和結構基礎。上述研究成果得到同行評審專家和雜誌編輯的高度讚賞，已於2007年5月18日在線發表於《The  Journal  of  Biological  Chemistry》雜誌。

    研究者們解析了全長ScNMT與豆蔻醯輔酶A的二元複合物的晶體結構，清晰地確定了以往報導中從未觀察到的ScNMT的N末端區域的結構，該區域緊靠酶催化反應中心，參與了豆蔻醯輔酶A和底物的識別和結合。研究者們進一步構建了一系列含有重要胺基酸點突變和截短形式的ScNMT突變體，運用生物化學的方法測定了它們的酶催化反應的各項動力學參數。基於結構生物學和生物化學的分析結果，研究者們揭示了NMT的N端區域參與底物和輔酶A的識別與結合，但並不影響最終的酶促反應活性。同時，這些研究結果還澄清了文獻報導中關於其它結構元素和胺基酸參與底物識別和結合功能的預測和假設。

    另外，研究者們還解析了全長ScNMT與豆蔻醯輔酶A和兩種高活性抑制劑形成的三元複合物的晶體結構，確定了抑制劑佔據著底物多肽的結合位點，從而發揮抑制酶活的功能。通過對抑制劑與酶相互作用的分析、以及與其它結構的比較，研究者們發現多肽底物的結合口袋具有較高的剛性，但部分胺基酸殘基的側鏈會根據抑制劑的化學性質和結構的不同發生微小的構象調整。據此，研究者們提出了對現有的抑制劑進行化學修飾和結構改造的思路，以增加它們與酶的相互作用和特異性。這些結果為研究和開發具有更高結合能力和抑制活性的抗真菌藥物提供了很好的結構基礎和理論指導。（引自生化細胞所）

 

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: [e-mail]

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.