Abstract

Phenylacetone monooxygenase from Thermobifida fusca (EC 1.14.13.92, PAMO) belongs to the Baeyer–Villiger family of monooxygenases and catalyzes the oxidation of various aromatic ketones to the corresponding esters using NADPH as a cofactor. In this study we analyzed the structure of the cofactor binding site, selected the most important amino acid residues for recognition of the phosphate group of the cofactor, and simulated enzyme structures with amino acid substitutions that could potentially lead to a change in the coenzyme specificity of the enzyme from NADPH to NADH. Based on the modeling, the most promising amino acid substitutions, T218D, T218E, K336A, and K336R, were proposed. Using site-directed mutagenesis we obtained genetic constructs containing genes encoding PAMOs with the corresponding amino acid substitutions, and the enzymes were expressed and purified. The resulting mutant PAMOs are able to bind NADH, but lack the ability to catalyze the oxidation of benzylacetone in the presence of NADH and show a deterioration of the Michaelis constants for NADPH. The catalytic constants of the mutant enzymes studied decrease slightly, and remain within the allowed experimental error.

中文翻译：

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定点诱变研究深红双歧杆菌苯丙酮单加氧酶的辅酶特异性机制

摘要

来自Thermobifida fusca的苯丙酮单加氧酶(EC 1.14.13.92, PAMO) 属于单加氧酶的 Baeyer-Villiger 家族，并使用 NADPH 作为辅因子催化各种芳香酮氧化成相应的酯。在这项研究中，我们分析了辅因子结合位点的结构，选择了最重要的氨基酸残基来识别辅因子的磷酸基团，并模拟了具有可能导致辅酶特异性改变的氨基酸取代的酶结构。从NADPH到NADH的酶。基于该模型，提出了最有希望的氨基酸取代 T218D、T218E、K336A 和 K336R。使用定点诱变，我们获得了包含编码 PAMOs 的基因的基因构建体，并具有相应的氨基酸取代，并且酶被表达和纯化。所得突变 PAMO 能够结合 NADH，但缺乏在 NADH 存在下催化苄基丙酮氧化的能力，并显示出 NADPH 的米氏常数恶化。所研究的突变酶的催化常数略有下降，并保持在允许的实验误差范围内。