Borbás Tímea, Zhang Jun, Cerny Matt A, Likó István, Cashman John R
Human BioMolecular Research Institute, San Diego, CA 92121, USA.
Drug Metab Dispos. 2006 Dec;34(12):1995-2002. doi: 10.1124/dmd.106.010827. Epub 2006 Sep 19.
To characterize the contribution of amino acid 360 to the functional activity of the human flavin-containing monooxygenase form 3 (FMO3) and form 1 (FMO1) in the oxygenation of drugs and chemicals, we expressed four FMO3 variants (i.e., Ala360-FMO3, His360-FMO3, Gln360-FMO3, and Pro360-FMO3) and one FMO1 variant (i.e., Pro360-FMO1) and compared them to wild-type enzymes (Leu360-FMO3 and His360-FMO1, respectively). The amino acid substitutions were introduced into wild-type FMO3 or FMO1 cDNA by site-directed mutagenesis. The thermal stability of variants of Leu360 FMO3 was also studied, and the thermal stability was significantly different from that of wild-type FMO3. The influence of different substrates to modulate the catalytic activity of FMO3 variants was also examined. Selective functional substrate activity was determined with mercaptoimidazole, chlorpromazine, and 10-[(N,N-dimethylaminopentyl)-2-(trifluoromethyl)]phenothiazine. Compared with wild-type FMO3, the Ala360-FMO3 and His360-FMO3 variants were less catalytically efficient for mercaptoimidazole S-oxygenation. N-Oxygenation of chlorpromazine was significantly less catalytically efficient for His360-FMO3 compared with wild-type FMO3. Human Pro360-FMO1 was significantly more catalytically efficient at S-oxygenating mercaptoimidazole and chlorpromazine compared with wild-type FMO1. The data support the mechanism that the Pro360 loci affect thermal stability of FMO3. Because different amino acids at position 360 affect substrate oxygenation in a unique fashion compared with that of FMO3 stimulation, we conclude that the mechanism of stimulation of FMO3 is distinct from that of enzyme catalysis. A molecular model of human FMO3 was also constructed to help explain the results. The increase in catalytic efficiency observed for Pro360 in human FMO3 was also observed when the His of FMO1 was replaced by Pro at loci 360.
为了阐明氨基酸360对人含黄素单加氧酶3型（FMO3）和1型（FMO1）在药物和化学物质氧化反应中的功能活性的贡献，我们表达了四种FMO3变体（即丙氨酸360 - FMO3、组氨酸360 - FMO3、谷氨酰胺360 - FMO3和脯氨酸360 - FMO3）和一种FMO1变体（即脯氨酸360 - FMO1），并将它们与野生型酶（分别为亮氨酸360 - FMO3和组氨酸360 - FMO1）进行比较。通过定点诱变将氨基酸替换引入野生型FMO3或FMO1 cDNA中。还研究了亮氨酸360 FMO3变体的热稳定性，其热稳定性与野生型FMO3显著不同。还检测了不同底物对调节FMO3变体催化活性的影响。用巯基咪唑、氯丙嗪和10 - [（N，N - 二甲基氨基戊基）- 2 - （三氟甲基）]吩噻嗪测定选择性功能底物活性。与野生型FMO3相比，丙氨酸360 - FMO3和组氨酸360 - FMO3变体对巯基咪唑S - 氧化的催化效率较低。与野生型FMO3相比，组氨酸360 - FMO3对氯丙嗪的N - 氧化催化效率显著降低。与野生型FMO1相比，人脯氨酸360 - FMO1在巯基咪唑和氯丙嗪的S - 氧化方面催化效率显著更高。数据支持脯氨酸360位点影响FMO3热稳定性的机制。由于360位的不同氨基酸与FMO3刺激相比以独特方式影响底物氧化，我们得出结论，FMO3的刺激机制与酶催化机制不同。还构建了人FMO3的分子模型以帮助解释结果。当FMO1的组氨酸在360位点被脯氨酸取代时，也观察到了人FMO3中脯氨酸360催化效率的增加。
