Structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoylCoA and peptide provide insights about substrate recognition and catalysis
Structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoylCoA and peptide provide insights about substrate recognition and catalysis
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DOI：
10.1021/bi0101401
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发表时间：
2001-05-29
期刊：
影响因子：
2.9
通讯作者：
Gordon, JI
中科院分区：
文献类型：
--
作者：
Farazi, TA;Waksman, G;Gordon, JI
肉豆蔻酰COA：蛋白质 N-肉豆蔻酰转移酶 (Nmt) 将肉豆蔻酸附着到参与多种信号转导级联和其他关键细胞功能的蛋白质的 N 末端甘氨酸残基上。为了深入了解底物识别和催化的结构基础，我们确定了酿酒酵母 Nmt1p 与肉豆蔻酰辅酶 A 的二元复合物的结构，分辨率为 2.2 埃，以及 Nmt1p 与不可水解的肉豆蔻酰辅酶 A 类似物 [S-(2- 氧代)十五酰辅酶 A] 和八肽底物 (GLYASKLA) 的三元复合物的结构，以2.5埃分辨率。该二元复合物揭示了肉豆蔻酰辅酶A如何改变酶的构象以促进肉豆蔻酰辅酶A和肽的结合，并将F170和L171的主链酰胺鉴定为氧阴离子孔，该氧阴离子孔使反应性硫酯羰基极化。三元复合物结构揭示了酶的肽结合特异性的细节，并阐明了其酰基转移的机制。 N 端甘氨酸铵的位置靠近蛋白质的 C 端羧酸盐，在那里它准备进行所需的去质子化为胺。在此构象中，亲核试剂距离硫酯羰基 6.3 埃。提出了一种催化机制，一旦去质子化开始，N-末端甘胺就可以通过沿着 Psi 旋转来接近硫酯羰基。 II 键网络促进了这种运动，并导致甘氨酸氮亲核试剂和羰基之间发生反应。硫与 CoA 腺苷胺的分子内 II 键合可能会促进 CoA 从四面体中间体的损失。这提供了一个紧凑的离去基团，并为 CoA 结构中观察到的弯曲提供了作用。底物 N 末端对甘氨酸的绝对要求可以通过其胺灵活旋转的要求来解释。
MyristoylCOA:protein N-myristoyltransferase (Nmt) attaches myristate to the N-terminal Gly residue of proteins involved in a variety of signal transduction cascades, and other critical cellular functions. To gain insight about the structural basis of substrate recognition and catalysis, we determined the structures of a binary complex of Saccharomyces cerevisiae Nmt1p with myristoylCoA to 2.2 Angstrom resolution and of a ternary complex of Nmt1p with a nonhydrolyzable myristoylCoA analogue [S-(2- oxo)pentadecylCoA] and an octapeptide substrate (GLYASKLA) to 2.5 Angstrom resolution. The binary complex reveals how myristoylCoA alters the conformation of the enzyme to promote binding of both myristoylCoA and peptide and identifies the backbone amides of F170 and L171 as an oxyanion hole which polarizes the reactive thioester carbonyl. The ternary complex structure reveals details of the enzyme's peptide binding specificity and illuminates its mechanism of acyl transfer. The N-terminal Gly ammonium is positioned in close proximity to the C-terminal carboxylate of the protein, where it is poised to undergo the required deprotonation to an amine. In this conformation, the nucleophile is 6.3 Angstrom away from the thioester carbonyl. A catalytic mechanism is proposed whereby, once deprotonation is initiated, the N-terminal Gly amine can approximate the thioester carbonyl by rotating along Psi. This motion is facilitated by a II-bond network and leads to reaction between the glycine nitrogen nucleophile and the carbonyl. Loss of CoA from the tetrahedral intermediate may be facilitated by intramolecular II-bonding of the sulfur to the adenylamine of CoA. This affords a compact leaving group and lends a role for the observed bends in the CoA structure. The absolute requirement for Gly at the N-terminus of substrates is explained by the requirement for flexible rotation of its amine.