Directed evolution of the substrate specificity of dialkylglycine decarboxylase.
Directed evolution of the substrate specificity of dialkylglycine decarboxylase.
复制标题
二烷基甘氨酸脱羧酶底物特异性的定向进化。
DOI：
10.1016/j.bbapap.2014.12.003
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Toney,MichaelD
中科院分区：
文献类型：
--
作者：
Taylor,JaredL;Price,JosephE;Toney,MichaelD
二烷基甘氨酸脱羧酶（DGD）是一种特殊的磷酸吡哆醛依赖酶，在其乒乓催化循环的第一个反应中催化脱羧，在第二个半反应中催化转氨化。定向进化将DGD的底物特异性从2-氨基异丁酸酯（AIB）改变为1-氨基环己烷-1-羧酸酯（AC6C）。四轮定向进化导致了几个突变体的鉴定，最后一轮的克隆包含五个持续突变。最佳无性系对AC6C的催化效率提高了约5倍，kcat降低了约2.5倍，kcat增加了约2倍。另外几轮定向进化并没有提高对AC6C的催化活性。五个持久突变中只有一个（S306F）接近活性位点。除1个克隆外，在所有33个克隆中都观察到S306F，并且该突变被证明可以稳定酶的变性。另外四个持久突变位于酶的表面附近。S306F突变和远端突变均对AIB和AC6C的动力学参数有显著影响。分子动力学模拟表明，突变改变了酶的构象景观，倾向于更开放的活性位点构象，促进了更大底物的反应性。我们推测AC6C的kcat/ km的小幅增加是由于两个限制因素。首先是通过协同脱羧/质子转移过渡态催化氧化脱羧的机制要求。二是在乒乓催化循环的后半反应中，DGD必须在相同的活性位点催化转氨化。
Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~ 2.5-fold decrease in KMand ~ 2-fold increase in kcat, giving a modest ~ 5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KMfor AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.