Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.
Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.
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DOI：
10.1038/srep26628
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发表时间：
2016-05-26
影响因子：
4.6
通讯作者：
Munro AW
中科院分区：
文献类型：
--
作者：
Chenge J;Kavanagh ME;Driscoll MD;McLean KJ;Young DB;Cortes T;Matak-Vinkovic D;Levy CW;Rigby SE;Leys D;Abell C;Munro AW
结核分枝杆菌（Mtb）引起结核病（TB）。毒力强的Mtb H37Rv菌株编码20种细胞色素P450 （CYP）酶，其中许多与Mtb在人类宿主中的存活和致病性有关。生物信息学分析显示，CYP144A1仅在分枝杆菌属中保留，特别是在引起人类和动物疾病的物种中。转录组学注释揭示了两个可能的CYP144A1起始密码子，导致(i)“全长”434个氨基酸版本（CYP144A1- flv）和（ii）“截断”404个氨基酸版本（CYP144A1- trv）的表达。计算分析预测CYP144A1-FLV扩展的n端区域大部分是非结构化的。在血红素结合状态下纯化了CYP144A1 FLV和TRV形式。质谱分析证实了完整的、his6标记的CYP144A1-FLV和-TRV的产生，EPR显示在这两种情况下，半胱氨酸硫酸盐与血红素铁配合。水动力学分析表明，两种CYP144A1形式都是单体的。对CYP144A1-TRV进行结晶，确定了CYP144家族P450蛋白的第一个结构。CYP144A1-TRV具有开放的底物结合结构，具有较大的活性位点空腔。我们的数据首次证明，Mtb从不同的转录本中产生两种不同形式的CYP144A1，其中CYP144A1- trv是由缺乏5 ' -未翻译区和Shine-Dalgarno核糖体结合位点的无领导转录本产生的。
Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a “full-length” 434 amino acid version (CYP144A1-FLV) and (ii) a “truncated” 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5′-untranslated region and Shine-Dalgarno ribosome binding site.