Program in Gene Expression and Regulation, The Wistar Institute, Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 2013 Sep 3;110(36):14652-7. doi: 10.1073/pnas.1310365110. Epub 2013 Aug 19.
Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity. Interestingly, these two NATs use different catalytic strategies to accomplish substrate-specific acetylation. In archaea, where this modification is less prevalent, only one NAT enzyme has been identified. Surprisingly, this enzyme is able to acetylate NatA and NatE substrates and is believed to represent an ancestral NAT variant from which the eukaryotic NAT machinery evolved. To gain insight into the evolution of NAT enzymes, we determined the X-ray crystal structure of an archaeal NAT from Sulfolobus solfataricus (ssNAT). Through the use of mutagenesis and kinetic analysis, we show that the active site of ssNAT represents a hybrid of the NatA and NatE active sites, and we highlight features of this protein that allow it to facilitate catalysis of distinct substrates through different catalytic strategies, which is a unique characteristic of this enzyme. Taken together, the structural and biochemical data presented here have implications for the evolution of eukaryotic NAT enzymes and the substrate specificities therein.
氨基末端乙酰化是真核生物中普遍存在的一种修饰，涉及越来越多的生物学过程。有六种已知的真核生物氨基末端乙酰转移酶（NATs），它们在底物特异性的基础上彼此区分。迄今为止，已经对两种真核生物 NATs（NatA 和 NatE）进行了结构表征，其中 NatA 可乙酰化许多非甲硫氨酸氨基末端残基底物的α-氨基基团，如丝氨酸；NatE 活性需要底物氨基末端甲硫氨酸残基。有趣的是，这两种 NAT 采用不同的催化策略来完成底物特异性乙酰化。在修饰程度较低的古菌中，只鉴定出一种 NAT 酶。令人惊讶的是，这种酶能够乙酰化 NatA 和 NatE 底物，并且被认为代表了真核 NAT 机制进化的祖先 NAT 变体。为了深入了解 NAT 酶的进化，我们确定了来自 Sulfolobus solfataricus 的古菌 NAT 的 X 射线晶体结构（ssNAT）。通过使用诱变和动力学分析，我们表明 ssNAT 的活性位点代表了 NatA 和 NatE 活性位点的混合体，并且我们强调了该蛋白质的特征，该特征使其能够通过不同的催化策略促进不同底物的催化，这是该酶的独特特征。总之，这里提出的结构和生化数据对真核生物 NAT 酶的进化及其内在的底物特异性具有重要意义。
