Rothman Steven C, Kirsch Jack F
Department of Molecular and Cell Biology, University of California, Berkeley, 229 Stanley Hall #3206, Berkeley, CA 94720-3206, USA.
J Mol Biol. 2003 Mar 28;327(3):593-608. doi: 10.1016/s0022-2836(03)00095-0.
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 k(cat)/K(m)(Phe) and 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<or=p<or=0.008) concentration of mutations occurs in a subset of positions (set AAT-TAT) that is conserved (>or=75% identical) in AATases and variable (<75% identical) in TATases. Very few mutations occur in the intersection (set AAT intersection 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 15 A 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.
天冬氨酸转氨酶（AATase）和酪氨酸转氨酶（TATase）是大肠杆菌的旁系同源物，它们的序列同一性为43%。一个合理的模型假定TATase起源于一种祖先类AATase酶的复制。为了将进化后的AATase变体与同源TATase进行比较，对AATase进行了定向进化，使其成为具有TATase活性的酶。经过八轮DNA改组和体内筛选，随后与野生型AATase回交，产生的酶在k(cat)/K(m)(Phe)上提高了100 - 270倍，其酪氨酸转氨酶活性高达野生型大肠杆菌TATase的11%。将第7轮和第8轮的11个克隆中的氨基酸替换与AATase和TATase中的保守残基进行了比较。通过使用维恩图和集合论符号，可以方便且简洁地说明这些发现。在AATase中保守（≥75%相同）而在TATase中可变（<75%相同）的一个位置子集（集合AAT - TAT）中，出现了具有统计学意义（0.001≤p≤0.008）的突变浓度。在两种酶类型中都保守的氨基酸残基的交集（集合AAT交集TAT）中发生的突变很少。通过定点诱变将来自集合AAT - TAT的7个突变组合起来，得到一个构建体，其活性是第8轮最佳酶（有13个氨基酸替换）的60%。维恩图可能是一种普遍有用的工具，用于突出合理重新设计中最重要的特异性决定因素。将氨基酸替换映射到一种设计的TATase的氢化肉桂酸复合物的晶体结构上。在进化克隆中最常被替换的7个位置中有5个位于苯侧链的15埃范围内，但在那些发生过一两次突变的48个位置中只有6个在该半径范围内。上下文依赖性、中性突变、不同的选择压力和随机成分解释了这样一个观察结果：在直接进化的酶中发现的许多替换与现代天然TATase中发现的相应氨基酸不同。
