Hatakeyama Mayumi, Kitaoka Takuya, Ichinose Hirofumi
Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
FEMS Microbiol Lett. 2017 Jun 15;364(11). doi: 10.1093/femsle/fnx107.
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 Km values of wild-type and CYP57B3-mut were 14.6 × 103 ± 2.7 × 103 μM and 15.7 ± 1.0 μM, 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.
米曲霉细胞色素P450单加氧酶（CYP57B3）能够催化染料木黄酮的羟基化反应生成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，具有多种药理活性，CYP57B3有望作为一种生物催化剂应用于制药领域。因此，我们对CYP57B3进行了随机诱变，以提高其对染料木黄酮的催化活性。通过易错PCR将随机突变引入CYP57B3，构建了一个突变体文库。从2000个突变体中，我们分离出一个CYP57B3发生三重突变的突变体，导致三个氨基酸替换（V138I、S243N和V463F）。这种改变后的蛋白命名为CYP57B3-mut，从染料木黄酮生成3΄-羟基染料木黄酮具有较高的活性。在生物转化反应中，表达CYP57B3-mut的酿酒酵母产生3΄-羟基染料木黄酮的量比野生型CYP57B3高14倍。体外动力学分析表明，野生型和CYP57B3-mut的Km值分别为14.6×103±2.7×103μM和15.7±1.0μM。这些结果表明，CYP57B3中V138、S243和V463的改变可显著提高其对染料木黄酮的亲和力。
