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首页> 外文期刊>African Journal of Biotechnology >Biochemical and kinetic characterization of geranylgeraniol 18-hydroxylase (CYP97C27) from Croton stellatopilosus Ohba
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Biochemical and kinetic characterization of geranylgeraniol 18-hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

机译：Croton stellatopilosus Ohba的香叶基香叶醇18-羟化酶（CYP97C27）的生化和动力学表征

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摘要

Geranylgeraniol 18-hydroxylase (EC 1.14.13.110) that exists solely in Croton stellatopilosus Ohba catalyses the last committed step of plaunotol biosynthetic pathways by conversion of geranylgeraniol (GGOH) to plaunotol. This enzyme and its gene are an attractive target for development of plaunotol production and its detailed biochemical properties need to be understood.?? Recently, even though the gene (CYP97C27) coding for GGOH 18-hydroxylase has been identified, cloned, and expressed in Escherichia coli system, the enzyme activity has been detected mainly in the insoluble fraction (20,000 g). This means that biochemical and kinetic studies could not be undertaken. However, our previous study indicated that this enzyme activity was easily and specifically detected in the microsomal fraction (100,000 g) of a crude enzyme extract. Therefore, in this report we describe a comprehensive biochemical characterization of GGOH 18-hydroxylase activity in the microsomal fraction from C. stellatopilosus Ohba. The oxygen-dependent enzyme activity of GGOH 18-hydroxylase was inhibited by carbon monoxide and the inhibition was partially reversible upon illumination with white light. Kinetic studies of the GGOH 18-hydroxylase showed high affinity to GGOH and NADPH with apparent Km values of 0.8 and 53 μM, respectively. Furthermore, the enzyme activity was inhibited by P450 inhibitors, including ancymidol, metyrapone, miconazole, potassium cyanide and cytochrome c, with the IC50 values of 428, 65, 75, 66 and 8 μM, respectively. Based on the biochemical and kinetic characteristics, the GGOH 18-hydroxylase in the microsomal fraction is likely a P450 encoded by CYP97C27 gene as previously described.
机译：仅存在于巴豆金缕梅中的香叶基香叶醇18-羟化酶（EC 1.14.13.110）可通过将香叶基香叶醇（GGOH）转化为普洛诺醇来催化Plaunotol生物合成途径的最后一个固定步骤。该酶及其基因是开发青草醇的诱人靶标，需要详细了解其生化特性。近来，即使已鉴定，克隆并在大肠杆菌系统中表达了编码GGOH 18-羟化酶的基因（CYP97C27），该酶活性仍主要在不溶级分（20,000 g）中检测到。这意味着无法进行生化和动力学研究。但是，我们先前的研究表明，在粗酶提取物的微粒体级分（100,000 g）中，可以轻松，特异性地检测到这种酶的活性。因此，在本报告中，我们描述了来自C. stellatopilosus Ohba的微粒体级分中GGOH 18-羟化酶活性的全面生化特征。 GGOH 18-羟化酶的氧依赖性酶活性被一氧化碳抑制，并且该抑制在白光照射下是部分可逆的。 GGOH 18-羟化酶的动力学研究表明，其对GGOH和NADPH的亲和力很高，表观Km值分别为0.8和53μM。此外，酶活性受到P450抑制剂的抑制，包括ancymidol，甲吡酮，咪康唑，氰化钾和细胞色素c，IC50值分别为428、65、75、66和8μM。基于生物化学和动力学特性，微粒体级分中的GGOH 18-羟化酶可能是由CYP97C27基因编码的P450，如先前所述。

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