CYP51 from Trypanosoma brucei is obtusifoliol-specific
CYP51 from Trypanosoma brucei is obtusifoliol-specific
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DOI：
10.1021/bi048967t
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发表时间：
2004-08-24
期刊：
影响因子：
2.9
通讯作者：
Waterman, MR
中科院分区：
文献类型：
--
作者：
Lepesheva, GI;Nes, WD;Waterman, MR
CYP51（甾醇 14α-去甲基酶）的新亚型是甾醇生物合成中的必需酶，也是唑类抗真菌药物的主要靶标，在致病性原生生物、布氏锥虫 (TB)、间日锥虫、克氏锥虫和大型利什曼原虫中发现。这些序列与其他生物界的甾醇 14α-脱甲基酶具有 80% 的氨基酸同一性和 25% 的同一性。与来自结核分枝杆菌 (MT) 的 CYP51 的 BC 环和螺旋 F 和 G 对齐的 CYP51 家族其余部分中保守的残基的差异意味着原生动物酶活性位点腔的拓扑结构可能发生变化。克隆来自结核病的 CYP51 和细胞色素 P450 还原酶 (CPR)，在大肠杆菌中表达并纯化。 P450 具有正常的光谱特征（包括绝对吸光度、一氧化碳和配体结合光谱），可被 TB 和大鼠 CPR 有效降低，但与人 CYP51 相比，对三种测试的唑类抑制剂表现出改变的特异性，并且与人、白色念珠菌和 MT 亚型相反，显示出对 obtusifoliol 的深刻底物偏好（周转 5.6 min(-1)）。它与其他已知的 CYP51 底物相互作用较弱；缓慢的羊毛甾醇转化主要产生14α-甲醛中间体。尽管钝叶醇特异性是 CYP51 植物亚型的典型特征，但原生动物基因组中的一组甾醇生物合成酶以及有关动质体细胞甾醇组成的可用信息表明 TBCYP51 的底物偏好可能反映了锥虫科中一种新的甾醇生物合成途径。
New isoforms of CYP51 (sterol 14alpha-demethylase), an essential enzyme in sterol biosynthesis and primary target of azole antimycotic drugs, are found in pathogenic protists, Trypanosoma brucei (TB), T. vivax, T. cruzi, and Leishmania major. The sequences share similar to80% amino acid identity and are similar to25% identical to sterol 14alpha-demethylases from other biological kingdoms. Differences of residues conserved throughout the rest of the CYP51 family that align with the BC-loop and helices F and G of CYP51 from Mycobacterium tuberculosis (MT) imply possible alterations in the topology of the active site cavity of the protozoan enzymes. CYP51 and cytochrome P450 reductase (CPR) from TB were cloned, expressed in Escherichia coli, and purified. The P450 has normal spectral features (including absolute absorbance, carbon monoxide, and ligand binding spectra), is efficiently reduced by TB and rat CPR but demonstrates altered specificity in comparison with human CYP51 toward three tested azole inhibitors, and contrary to the human, Candida albicans, and MT isoforms, reveals profound substrate preference toward obtusifoliol (turnover 5.6 min(-1)). It weakly interacts with the other known CYP51 substrates; slow lanosterol conversion predominantly produces the 14alpha-carboxyaldehyde intermediate. Although obtusifoliol specificity is typical for plant isoforms of CYP51, the set of sterol biosynthetic enzymes in the protozoan genomes together with available information about sterol composition of kinetoplastid cells suggest that the substrate preference of TBCYP51 may reflect a novel sterol biosynthetic pathway in Trypanosomatidae.