Impacts of amino acid substitutions in fungal cytochrome P450 monooxygenase (CYP57B3) on the effective production of 3′-hydroxygenistein
Impacts of amino acid substitutions in fungal cytochrome P450 monooxygenase (CYP57B3) on the effective production of 3′-hydroxygenistein
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DOI：
10.1093/femsle/fnx107
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发表时间：
2017-06-01
影响因子：
2.1
通讯作者：
Ichinose, Hirofumi
中科院分区：
文献类型：
--
作者：
Hatakeyama, Mayumi;Kitaoka, Takuya;Ichinose, Hirofumi
米曲霉细胞色素 P450 单加氧酶 (CYP57B3) 能够催化染料木黄酮羟基化产生 3'-羟基染料木黄酮。由于染料木黄酮的羟基化衍生物（包括 3'-羟基染料木黄酮）表现出多种药理活性，因此 CYP57B3 可能可用作制药领域的生物催化剂。因此，我们对 CYP57B3 进行随机诱变，以提高其对金雀异黄酮的催化活性。通过易错 PCR 将随机突变引入 CYP57B3，从而构建突变体文库。我们从 2000 个突变体中分离出一个 CYP57B3 具有三重突变的突变体，导致三个氨基酸取代（V138I、S243N 和 V463F）。这种改变的蛋白质被命名为 CYP57B3-mut，对于从染料木黄酮生产 3'-羟基染料木黄酮具有高水平的活性。在生物转化反应中，表达 CYP57B3-mut 的酿酒酵母显示 3'-羟基染料木黄酮的产量比野生型 CYP57B3 高 14 倍。体外动力学分析显示野生型和CYP57B3-mut的K-m值分别为14.6×10(3)+/-2.7×10(3)μM和15.7+/-1.0μM。这些结果表明，CYP57B3 中 V138、S243 和 V463 的改变可以显着提高 CYP57B3 对金雀异黄酮的亲和力。
Aspergillus oryzae cytochrome P450 monooxygenase (CYP57B3) is capable of catalyzing hydroxylation of genistein to produce 3'-hydroxygenistein. Because hydroxylated derivatives of genistein, including 3'-hydroxygenistein, exhibit various pharmacological activities, CYP57B3 would potentially be useful as a biocatalyst in the pharmaceutical field. We therefore performed random mutagenesis of CYP57B3 to improve its catalytic activities for genistein. Random mutations were introduced by error-prone PCR into CYP57B3, resulting in construction of a library of mutants. From 2000 mutants, we isolated one with a triple mutation in CYP57B3, resulting in three amino acid substitutions (V138I, S243N and V463F). The altered protein, designated CYP57B3-mut, had a high level of activity for the production of 3'-hydroxygenistein from genistein. In bioconversion reactions, Saccharomyces cerevisiae expressing CYP57B3-mut showed 14-fold higher production of 3'-hydroxygenistein than that of wild-type CYP57B3. In vitro kinetic analysis revealed that K-m values of wild-type and CYP57B3-mut were 14.6 x 10(3) +/- 2.7 x 10(3) mu M and 15.7 +/- 1.0 mu M, respectively. These results suggest that the affinity of CYP57B3 for genistein could be dramatically improved by the alterations of V138, S243 and V463 in CYP57B3.