How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.
How does an enzyme evolved in vitro compare to naturally occurring homologs possessing the targeted function? Tyrosine aminotransferase from aspartate aminotransferase.
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DOI：
10.1016/s0022-2836(03)00095-0
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发表时间：
2003-03
影响因子：
5.6
通讯作者：
S. C. Rothman;J. Kirsch
中科院分区：
文献类型：
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作者：
S. C. Rothman;J. Kirsch
天冬氨酸氨基转移酶（AATase）和酪氨酸氨基转移酶（TATase）是大肠杆菌的旁系同源物，具有43%的序列同一性。一个合理的模型假设TATase是由一种祖先的AATase样酶的复制产生的。进行AAT酶向具有TAT酶活性的酶的定向进化，以将进化的AAT酶变体与同源的TAT酶进行比较。八轮DNA改组和体内选择，然后与WT AATase回交，产生的酶表现出100-270倍的kcat/KmPhe增加，并具有WT大肠杆菌TATase的酪氨酸转氨酶活性的11%。将来自第7轮和第8轮的11个克隆中的氨基酸取代与AAT酶和TAT酶中的保守残基进行比较。文氏图和集合论符号的使用，方便和复杂的说明的结果。统计学显著（0.001≤p≤0.008）的突变浓度发生在AAT酶中保守（≥75%相同）和TAT酶中可变（<75%相同）的位置子集（达特集合）中。在两种酶类型中保守的氨基酸残基的交叉点（集合AAT →达特）中发生的突变非常少。通过定点诱变将来自达特组的七个突变组合，得到活性为最佳第8轮酶的60%的构建体，其具有13个氨基酸置换。文氏图可以提供一个普遍有用的工具，突出最重要的特性决定因素，合理的重新设计。氨基酸置换映射到所设计的TAT酶的氢化肉桂酸复合物的晶体结构上。在进化的克隆体中，最常被取代的7个位置中有5个在苯基侧链的15 μ m范围内，但48个位置中只有6个被突变了一次或两次。上下文依赖性，中性突变，不同的选择压力，和随机成分提供了解释的观察，许多取代中发现的直接进化的酶不同于相应的氨基酸中发现的现代天然TATases。
Aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase) are Escherichia coli paralogs that share 43% sequence identity. A plausible model posits that TATase arose from a duplication of an ancestral AATase-like enzyme. Directed evolution of AATase to an enzyme having TATase activity was undertaken in order to compare the evolved AATase variants with homologous TATases. Eight rounds of DNA shuffling and in vivo selection followed by a backcross with WT AATase produced enzymes that exhibited 100–270-fold increases in kcat/KmPheand had as much as 11% of the tyrosine aminotransferase activity of WT E.coli TATase. Amino acid substitutions in 11 clones from rounds 7 and 8 were compared with conserved residues in AATases and TATases. The findings are conveniently and compactly illustrated by the use of Venn diagrams and set theory notation. A statistically significant (0.001≤p≤0.008) concentration of mutations occurs in a subset of positions (set AAT – TAT ) that is conserved (≥75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT ∩ TAT ) of amino acid residues that are conserved in both enzyme types. Seven mutations from set AAT – TAT were combined by site-directed mutagenesis to give a construct that is 60% as active as the best round 8 enzyme, which has 13 amino acid replacements. The Venn diagrams may provide a generally useful tool to highlight the most important specificity determinants for rational redesign. Amino acid replacements were mapped onto the crystal structure of a hydrocinnamate complex of a designed TATase. Five of the seven positions most frequently substituted in the evolved clones are within 15Å of the phenyl side-chain, but only six of the 48 positions that were mutated once or twice are within that radius. Context dependence, neutral mutations, different selective pressures, and stochastic components provide explanations for the observation that many of the substitutions found in the directly evolved enzymes differ from the corresponding amino acids found in the modern natural TATases.