在金屬單加氧酵素，如何去進行在不同基質的C-H鍵氧化機制與酵素內部如何調控位置(區域)特異性或立體選擇性，一直都是讓人非常感興趣的議題。巨大芽胞桿菌（Bacillus megaterium）的細胞色素P450，使用大氣中的氧氣當做氧化劑，氧化C12－C20脂肪酸在ω-1，ω-2或ω-3 C-H鍵的位置。我們利用基因工程進行定點突變細胞色素P450 BM3 (Cytochrome P450 BM-3)的實驗，以結合理想化設計與導引式演化的定點突變法，對於P450 BM3的胺基酸序列進行定點突變，嘗試從靠近活性中心周圍不同的氨基酸位置，篩選出扮演關鍵性角色的位置。
本研究中，除了利用3mt (BM3 A74G F87V L188Q)蛋白來研究其催化中長度的烷烴化合物基質外，亦藉由實驗定點突變Ala328Phe （F328），進行不同碳數直鏈烷類篩選，發現位置選擇性會從2- 3- 和4-醇的催化，轉變為偏向2-號位置為主的選擇性。基質選擇性則由長碳鏈脂肪酸（C12-C22）變成具有丁烷催化能力。Leu188Pro（P450 BM3-FP188）可控制氣體分子進入的位置，能增加丁烷轉換成2-丁醇的催化活性12.5%；當我們引入Ala74Glu（P450 BM3-FPE74），可以大幅提升丁烷轉換成2-丁醇的催化活性提升至將近40％。
最後，一系列氘化和氟化的烷烴基質做為探針，並藉由酵素氧化這些烷烴基質的產物深入了解C-H鍵氧化控制機制。針對各式含氘丁烷與含氟辛烷的單加氧之反應性、羥基化的位置選擇性以及產物的掌性分布做歸納，找出其調控的動態基礎。

The control oxidation of aliphatic C-H bonds in regio- and/or stereo-selective manner by metalloenzymes is of great interest to scientists. Cytochrome P450 BM3 from Bacillus megaterium oxidizes C12-C20 fatty acids at the ω-1, ω-2, or ω-3 position of the CH bond, respectively. We employ the molecular manipulation techniques to overexpress the recombinant BM3 strain and carry out its site-directed mutagenesis study for generation of a variety of strains towards variable selective oxidation with different substrates.
In my study, strain 3mt, A74G F87V L188Q is exploited for the controlled oxidations of medium-chain length alkanes. Single mutation variant, BM3 mutant Ala328Phe (F328), was found with its capability of selective oxidation at the ω1 position of C4-C10 straight-chain alkanes. Interestingly, long-chain fatty acids (C12-C20) are no longer its substrates. It provides a great base to allow us engineering P450 BM3 protein from normal alkanes with medium-chain length such as n-octane gradually to smaller alkane such as n-butane for subsequent studies of controlled oxidation. The introduction of the second mutation, at Leu188Pro (P450 BM3-FP188) can improve the catalytic efficiency from butane to 2-butanol for 12.5%. In addtion, introducinge Ala74Glu (P450 BM3-FPE74) can shrink the substrate pocket down to half of the volume and significantly enhance the catalytic activity of butane to 2- butanol for nearly 40%.
Typically, enzymes invoke host–guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. To further investigate this issue, we also develope a series of deuterated and fluorinated aliphatic substrates as probes to gain insights into the controlled C-H oxidations of hydrocarbons facilitated by these enzymes. The effects resulting from the introduction of deuterated butane (isotopomers) and fluorinated octane (Bioisostere) substituents on the mechanistic insights for C-H oxidation and the controls for regio-specificity and stereo-selectivity of the C-H bond activation are discussed.

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