Luanloet Thikumporn, Sucharitakul Jeerus, Chaiyen Pimchai
Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
FEBS J. 2015 Aug;282(16):3107-25. doi: 10.1111/febs.13220. Epub 2015 Feb 23.
2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the interaction between Tyr223 and the substrate is important for ensuring hydroxylation. These results highlight how the active site of a flavoenzyme is able to deal with the presence of multiple forms of a substrate in solution and ensure efficient hydroxylation.
来自假单胞菌属MA-1的2-甲基-3-羟基吡啶-5-羧酸（MHPC）加氧酶（EC 1.14.12.4）是一种黄素依赖性单加氧酶，可催化羟基化和芳香环裂解反应。利用定点诱变技术，结合配体结合、产物分析和瞬态动力学实验，对位于距MHPC约5埃处的两个残基Tyr223和Tyr82的功能作用进行了表征。Tyr223的突变导致酶变体的羟基化活性受损，其底物结合的Kd值比野生型酶大5至10倍。由于该残基与位于MHPC 3-羟基旁边的水分子相邻，结果表明Tyr223、H2O和MHPC的3-羟基之间的相互作用对于底物结合和羟基化很重要。相比之下，Tyr82His和Tyr82Phe变体的底物结合Kd与野生型酶相似。然而，在两个变体的反应中，只有约40%-50%的底物被羟基化，而在野生型酶反应中，大部分底物被羟基化。在游离溶液中，MHPC或5-羟基烟酸以单阴离子和三极离子形式的混合物存在，而只有三极离子形式与野生型酶结合。三极离子MHPC的结合将通过亲电芳香取代机制实现高效羟基化。对于Tyr82His和Tyr82Phe变体，两种形式的底物都可以与酶结合，这表明Tyr82处的突变消除了酶对三极离子形式的选择性。瞬态动力学研究表明，两个Tyr82变体的羟基化速率常数比野生型酶高约2至2.5倍。总之，我们的研究结果表明，Tyr82对于MHPC加氧酶对三极离子物种的结合选择性很重要，而Tyr223与底物之间的相互作用对于确保羟基化很重要。这些结果突出了黄素酶的活性位点如何能够处理溶液中多种形式底物的存在并确保高效羟基化。
