Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase
Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase
复制标题
DOI：
10.1128/aem.69.1.419-426.2003
复制
发表时间：
2003-01-01
影响因子：
4.4
通讯作者：
Janssen, DB
中科院分区：
文献类型：
--
作者：
Kamerbeek, NM;Olsthoorn, AJJ;Janssen, DB
来自荧光假单胞菌ACB的4-羟基苯乙酮单加氧酶（HAPMO）催化NADPH和氧依赖的4-羟基苯乙酮Baeyer-Villiger氧化生成相应的乙酸酯。使用重组大肠杆菌纯化的酶，我们发现广泛的羰基化合物在结构上或多或少与4-羟基苯乙酮相似，也是这种含havin的单加氧酶的底物。另一方面，作为其他Baeyer-Villiger单加氧酶（BVMOs）底物的一些羰基化合物不能被HAPMO转化。除了与芳香酮和醛进行Baeyer-Villiger反应外，该酶还能催化芳香硫化物的亚砜化反应。此外，一些杂环和脂肪羰基化合物也很容易被BVMO转化。为了探究HAPMO的对映选择性，研究了双环庚-2-烯-6- 1和两个芳基烷基硫化物的转化。单加氧酶优选转化(1R,5S)-双环庚-2-烯-6- 1，其对映体比(E)为20，从而能够进行动力学拆分得到（1S,5R）对映体。这两种对映体的完全转化导致两种区域异构体内酯的积累，得到的两种内酯的对映体过量（ee）为77%，(1S,5R)-2为34%，(1R,5S)-3为34%。使用甲基4-甲基硫醚和甲基苯基硫醚，我们发现HAPMO在不对称形成相应的(S)-亚砜（ee > 99%）方面是高效和高选择性的。这里描述的HAPMO的生物催化特性显示了这种酶在生物技术应用方面的潜力。
The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this Havin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.