Cytochrome P450 enzymes (P450) play essential roles in redox metabolism in all domains of life including detoxification reactions and sterol biosynthesis. The activity of P450s is fuelled by two electron-transferring mechanisms, heme-independent P450 reductase (CPR) and the heme-dependent cytochrome b₅ (CYB5)/cytochrome b₅ reductase (CB5R) system. In this study, we characterised the role and regulation of the cytochrome b₅ CybE in the fungal pathogen Aspergillus fumigatus. Deletion of the CybE encoding gene (cybE) caused a severe growth defect in two different A. fumigatus isolates, emphasising the importance of the CB5R system in this pathogen, while the non-essentiality of cybE indicates the partial redundancy of the CPR and CB5R systems. Interestingly, the growth defect caused by the cybE loss of function was even more drastic in A. fumigatus strain AfS77 compared to strain A1160P+ indicating a strain-dependent degree of compensation, which is supported by azole resistance studies. In agreement with CybE being important for the assistance of the ergosterol biosynthetic P450 Cyp51, deletion of cybE decreased resistance to the Cyp51-targeting antifungal voriconazole and caused accumulation of the ergosterol pathway intermediate eburicol. Northern analysis indicated that CybE deficiency leads to the compensatory transcriptional upregulation of Cyp51-encoding cyp51A and CPR-encoding cprA. Overexpression of cybE did not affect azole resistance suggesting that CybE activity is not rate limiting. Expression of cybE was found to be repressed during iron starvation by the iron-regulatory transcription factor HapX demonstrating iron dependence of CybE not only at the level of enzyme activity but also at the level of gene expression.

中文翻译：

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细胞色素 b5 CybE 受铁可用性的调节，对烟曲霉的唑类抗性至关重要

细胞色素 P450 酶 (P450) 在包括解毒反应和甾醇生物合成在内的所有生命领域的氧化还原代谢中发挥重要作用。P450s 的活性由两种电子传递机制推动，即不依赖血红素的 P450 还原酶 (CPR) 和依赖血红素的细胞色素b ₅ (CYB5)/细胞色素b ₅还原酶 (CB5R) 系统。在这项研究中，我们描述了细胞色素b ₅ CybE 在真菌病原体烟曲霉中的作用和调节。CybE 编码基因 ( cybE ) 的缺失导致两种不同的烟曲霉严重的生长缺陷。分离株，强调了 CB5R 系统在该病原体中的重要性，而 cybE 的非必要性表明 CPR 和 CB5R 系统的部分冗余。有趣的是，与菌株 A1160P+ 相比，由cybE功能丧失引起的生长缺陷在烟曲霉菌株 AfS77中更为严重，表明补偿程度取决于菌株，这得到了唑类抗性研究的支持。同意 CybE 对麦角甾醇生物合成 P450 Cyp51 的帮助很重要，删除cybE降低对 Cyp51 靶向抗真菌药物伏立康唑的耐药性并导致麦角甾醇途径中间体 eburicol 的积累。Northern 分析表明，CybE 缺乏导致编码Cyp51 的cyp51A和编码 CPR 的cprA的代偿性转录上调。cybE的过表达不影响唑类抗性，表明 CybE 活性不受速率限制。发现cybE的表达在铁缺乏期间被铁调节转录因子 HapX 抑制，这表明 CybE 的铁依赖性不仅在酶活性水平上，而且在基因表达水平上。