Expression and Characterization of CYP51, the Ancient Sterol 14-demethylase Activity for Cytochromes P450 (CYP), in the White-Rot Fungus Phanerochaete chrysosporium
Expression and Characterization of CYP51, the Ancient Sterol 14-demethylase Activity for Cytochromes P450 (CYP), in the White-Rot Fungus Phanerochaete chrysosporium
复制标题
DOI：
10.1007/s11745-008-3239-5
复制
发表时间：
2008-12-01
期刊：
影响因子：
1.9
通讯作者：
Kelly, Steven
中科院分区：
文献类型：
--
作者：
Warrilow, Andrew;Ugochukwu, Cynthia;Kelly, Steven
黄孢原毛平革菌(Phanerochaete Chrysosporium)是第一个在其基因组中含有超过100个细胞色素P450(Cyp)基因的真菌基因组(类似于150)。它可以代谢木质纤维素和广泛的环境外来物质，包括许多致癌物质和污染物，其中可能涉及细胞色素P450。在本论文中，我们描述了从该生物中异源表达和鉴定的一种祖先CyP形式--甾醇14α-脱甲基酶(CyP51-EC1.14.13.70)。从cDNA文库中克隆了CYP51基因，并在对唑类抗真菌药物有较高亲和力的大肠杆菌和酿酒酵母中进行了表达。通过对酵母CYP51的条件敲除突变体的补充，观察到了功能的证明。该基因全长1,956个碱基，由7个外显子和6个内含子组成，编码550个氨基酸(62 KDa)的多肽。该蛋白对唑类抗真菌化合物具有较高的亲和力(k(D)0.25~0.45mU)。
Phanerochaete chrysosporium was the first fungal genome to exhibit more than one hundred cytochrome P450 (CYP) genes for a fungus within its genome (similar to 150). It can metabolize lignocellulose and a wide range of environmental xenobiotics including many carcinogens and pollutants where cytochromes P450 may be involved. In the present paper we describe the heterologous expression and characterization of an ancestral CYP form, sterol 14 alpha-demethylase (CYP51-EC1.14.13.70), from this organism. CYP51 was cloned from a cDNA library and expressed in both Escherichia coli, where it exhibited high affinity for azole antifungals, and Saccharomyces cerevisiae. Proof of function was observed by complementation of a conditional knock-down mutant of yeast CYP51. The CYP51 gene was found to be 1,956 bases long and contained 7 exons and 6 introns coding for a polypeptide 550 amino acids long (62 kDa). The CYP51 protein exhibited high affinity (k (d) 0.25-0.45 mu M) for azole antifungal compounds.