Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
J Biosci Bioeng. 2009 Sep;108(3):184-9. doi: 10.1016/j.jbiosc.2009.04.002.
The objective of this study was to enhance l-tryptophan hydroxylation activity of l-phenylalanine 4-hydroxylase. It had been known that l-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert l-tryptophan to 5-hydroxy-l-tryptophan and l-phenylalanine to l-tyrosine; however, the activity for l-tryptophan was extremely low compared to l-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceuml-phenylalanine 4-hydroxylase with high l-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L101 and W180 in C. violaceuml-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-l-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L101 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest l-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that k(cat) values for l-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s(-1) (W180F). In addition, the double mutant (L101Y-W180F) displayed higher l-tryptophan hydroxylation activity than the wild-type and the W180F and L101Y mutants. The k(cat) value of L101Y-W180F increased to 2.08 s(-1), showing a 5.2-fold increase compared to wild-type enzyme levels.
本研究旨在提高 l-色氨酸羟化酶对 l-苯丙氨酸 4-羟化酶的活性。已知紫色色杆菌的 l-苯丙氨酸 4-羟化酶可以将 l-色氨酸转化为 5-羟基-l-色氨酸和 l-苯丙氨酸转化为 l-酪氨酸；然而，与 l-苯丙氨酸的活性相比，l-色氨酸的活性极低。我们利用芳香族氨基酸羟化酶的晶体结构信息，生成了具有高 l-色氨酸羟化活性的紫色色杆菌 l-苯丙氨酸 4-羟化酶。计算机结构建模分析表明，在紫色色杆菌 l-苯丙氨酸 4-羟化酶中，L101 和 W180 处与底物和辅因子的疏水和/或堆积相互作用将增加羟化活性。基于这一假设，我们针对这些位点进行了饱和诱变，然后使用改良的 Gibbs 测定法评估 5-羟基-l-色氨酸的产率。从 L101 和 W180 突变文库中分别获得了 3 个和 9 个阳性突变体。在突变体中，L101Y 和 W180F 在各自的残基上显示出最高的 l-色氨酸羟化活性。稳态动力学分析表明，l-色氨酸羟化的 k(cat)值分别从野生型的 0.40 提高到 L101Y 的 1.02 和 W180F 的 0.51 s(-1)。此外，双突变体（L101Y-W180F）的 l-色氨酸羟化活性高于野生型和 W180F 和 L101Y 突变体。L101Y-W180F 的 k(cat)值增加到 2.08 s(-1)，与野生型酶水平相比提高了 5.2 倍。
