Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA.
Biochem Pharmacol. 2012 Apr 1;83(7):962-8. doi: 10.1016/j.bcp.2012.01.006. Epub 2012 Jan 13.
Flavin-containing monooxygenase (FMO) 5 belongs to a family of enzymes that catalyze the oxygenation of nucleophilic N- and S-containing compounds. The FMO enzyme family consists of five forms (FMOs1-5) that share about 50-60% sequence identity to each other. A comparison of FMOs showed that the pH-dependence profile for functional activity of FMO5 differed significantly from that of other FMO enzymes. The objective of this study was to examine the pH-dependence of FMO5 to gain insight into the mechanism of action of FMO5. Recombinant mouse and human FMO5 (mFMO5 and hFMO5, respectively) were expressed as maltose-binding fusion proteins from Escherichia coli, purified with affinity chromatography, and examined for their N-oxygenation functional activity at different pH values. hFMO5 showed a broader range and greater functional activity from pH 6 to 11 compared to mFMO5. mFMO5 lost almost all functional activity at pH 6, while hFMO5 maintained almost normal enzyme activity. In order to identify the amino acid residues involved in the effects of pH on hFMO5 and mFMO5 functional enzyme activity, pH-studies in the range of pH 6-9 were done with chimeras of recombinant mouse and human FMO5 and variants of both. Results of these studies and molecular modeling showed that residues responsible for the differences in the pH profile between mFMO5 and hFMO5 were located at positions 227 and 228 of the enzyme. Further variants were made to investigate the role of these amino acids. The results of this study may help to explain the mechanism of FMO function.
黄素单加氧酶（FMO）5 属于一类能够催化亲核 N 和 S 化合物氧化的酶。FMO 酶家族由五种形式（FMOs1-5）组成，它们彼此之间的序列同一性约为 50-60%。FMO 的比较表明，FMO5 功能活性的 pH 依赖性谱与其他 FMO 酶显著不同。本研究的目的是研究 FMO5 的 pH 依赖性，以深入了解 FMO5 的作用机制。重组的小鼠和人 FMO5（mFMO5 和 hFMO5，分别）分别从大肠杆菌中表达为麦芽糖结合融合蛋白，通过亲和层析纯化，并在不同 pH 值下检测其 N-氧化功能活性。与 mFMO5 相比，hFMO5 在 pH 6 到 11 的范围内具有更广泛的范围和更高的功能活性。mFMO5 在 pH 6 时几乎失去了所有的功能活性，而 hFMO5 则保持了几乎正常的酶活性。为了确定影响 pH 值对 hFMO5 和 mFMO5 功能酶活性的氨基酸残基，对重组小鼠和人 FMO5 的嵌合体以及两者的变体进行了 pH 范围在 6-9 的 pH 值研究。这些研究和分子建模的结果表明，导致 mFMO5 和 hFMO5 之间 pH 谱差异的残基位于酶的 227 和 228 位。进一步制作了变体以研究这些氨基酸的作用。本研究的结果可能有助于解释 FMO 功能的机制。
