生物谷：豆蔻酰辅酶A：蛋白质N-豆蔻酰转移酶（NMT）负责催化将豆蔻酰辅酶A上的豆蔻酸共价连接到真核生物和病毒的众多新生蛋白N末端的甘氨酸上，在多种重要细胞过程中发挥重要的生物学功能。遗传和生化研究结果表明NMT是抗真菌药物的理想靶标。在过去的十年中多个实验室对两个种属的NMTs进行了比较深入的结构和功能研究，但在获得的晶体结构中由于NMT的N端区域的缺失或无法定位，其生物学功能还不清楚。
生化与细胞所丁建平研究组与美国杜邦公司Stine Haskell研究中心Steven Gutteridge研究组经过三年多的合作，运用结构生物学和生物化学的手段阐明了酵母NMT（ScNMT）的N端区域的生物学功能和酶催化反应的分子机制，为设计和研制更加有效的抗真菌药物提供了有价值的生物学信息和结构基础。上述研究成果得到同行评审专家和杂志编辑的高度赞赏，已于2007年5月18日在线发表于《The Journal of Biological Chemistry》杂志。
研究者们解析了全长ScNMT与豆蔻酰辅酶A的二元复合物的晶体结构，清晰地确定了以往报道中从未观察到的ScNMT的N末端区域的结构，该区域紧靠酶催化反应中心，参与了豆蔻酰辅酶A和底物的识别和结合。研究者们进一步构建了一系列含有重要氨基酸点突变和截短形式的ScNMT突变体，运用生物化学的方法测定了它们的酶催化反应的各项动力学参数。基于结构生物学和生物化学的分析结果，研究者们揭示了NMT的N端区域参与底物和辅酶A的识别与结合，但并不影响最终的酶促反应活性。同时，这些研究结果还澄清了文献报道中关于其它结构元素和氨基酸参与底物识别和结合功能的预测和假设。
另外，研究者们还解析了全长ScNMT与豆蔻酰辅酶A和两种高活性抑制剂形成的三元复合物的晶体结构，确定了抑制剂占据着底物多肽的结合位点，从而发挥抑制酶活的功能。通过对抑制剂与酶相互作用的分析、以及与其它结构的比较，研究者们发现多肽底物的结合口袋具有较高的刚性，但部分氨基酸残基的侧链会根据抑制剂的化学性质和结构的不同发生微小的构象调整。据此，研究者们提出了对现有的抑制剂进行化学修饰和结构改造的思路，以增加它们与酶的相互作用和特异性。这些结果为研究和开发具有更高结合能力和抑制活性的抗真菌药物提供了很好的结构基础和理论指导。（引自生化细胞所）
原始出处:
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: jpding@sibs.ac.cn
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.
