Tolbert William D, Zhang Yang, Cottet Sarah E, Bennett Eric M, Ekstrom Jennifer L, Pegg Anthony E, Ealick Steven E
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Biochemistry. 2003 Mar 4;42(8):2386-95. doi: 10.1021/bi0268854.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes the formation of the aminopropyl group donor in the biosynthesis of the polyamines spermidine and spermine. The enzyme is synthesized as a protein precursor and is activated by an autocatalytic serinolysis reaction that creates the pyruvoyl group. The autoprocessing reaction proceeds via an N --> O acyl rearrangement, generating first an oxyoxazolidine anion intermediate followed by an ester intermediate. A similar strategy is utilized in self-catalyzed protein splicing reactions and in autoproteolytic activation of protein precursors. Mutation of Ser68 to alanine in human AdoMetDC prevents processing by removing the serine side chain necessary for nucleophilic attack at the adjacent carbonyl carbon atom. We have determined the X-ray structure of the S68A mutant and have constructed models of the proenzyme and the oxyoxazolidine intermediate. Formation of the oxyoxazolidine intermediate is promoted by a hydrogen bond from Cys82 and stabilized by a hydrogen bond from Ser229. These observations are consistent with mutagenesis studies, which show that the C82S and C82A mutants process slowly and that the S229A mutant does not process at all. Donation of a proton by His243 to the nitrogen atom of the oxyoxazolidine ring converts the oxyoxazolidine anion to the ester intermediate. The absence of a base to activate the hydroxyl group of Ser68 suggests that strain may play a role in the cleavage reaction. Comparison of AdoMetDC with other self-processing proteins shows no common structural features. Comparison to histidine decarboxylase and aspartate decarboxylase shows that these pyruvoyl-dependent enzymes evolved different catalytic strategies for forming the same cofactor.
S-腺苷甲硫氨酸脱羧酶（AdoMetDC）是一种依赖于丙酮酸的酶，它在多胺亚精胺和精胺的生物合成中催化氨丙基供体的形成。该酶以蛋白质前体的形式合成，并通过产生丙酮酸基团的自催化丝氨酸水解反应被激活。自加工反应通过N→O酰基重排进行，首先生成一个氧代恶唑烷阴离子中间体，随后生成一个酯中间体。类似的策略也用于自催化蛋白质剪接反应和蛋白质前体的自蛋白水解激活。人类AdoMetDC中Ser68突变为丙氨酸会通过去除亲核攻击相邻羰基碳原子所需的丝氨酸侧链来阻止加工。我们已经确定了S68A突变体的X射线结构，并构建了酶原和氧代恶唑烷中间体的模型。氧代恶唑烷中间体的形成由Cys82的氢键促进，并由Ser229的氢键稳定。这些观察结果与诱变研究一致，诱变研究表明C82S和C82A突变体加工缓慢，而S229A突变体根本不进行加工。His243向氧代恶唑烷环的氮原子提供一个质子，将氧代恶唑烷阴离子转化为酯中间体。缺乏激活Ser68羟基的碱表明应变可能在裂解反应中起作用。将AdoMetDC与其他自加工蛋白进行比较，未发现共同的结构特征。与组氨酸脱羧酶和天冬氨酸脱羧酶的比较表明，这些依赖于丙酮酸的酶为形成相同的辅因子进化出了不同的催化策略。
