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首页> 外文期刊>The Journal of Antibiotics: An International Journal >Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392.
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Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392.

机译：P450Um-1（一种新型细菌P450基因）的克隆和异源表达，用于免疫抑制剂AS1387392的羟基化。

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摘要

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.
机译：涉及通过诸如微生物之类的生物酶对原始底物进行酶促修饰的生物转化技术是改善基础化合物的药代动力学或物理化学性质的有价值的工具。真菌代谢物AS1387392是一种组蛋白脱乙酰基酶抑制剂，有潜力用作治疗性免疫抑制剂。但是，它缺乏合成衍生物所必需的官能团是一个缺点。 Amycolatopsis azurea JCM-3275催化了AS1387392到AS1429716的羟基化，这可以通过AS1429716中存在的额外羟基部分促进更多衍生物的合成。该反应被细胞色素P450抑制剂美拉酮抑制，表明细胞色素P450可能是转化的原因。随后构建简并的PCR引物，并用于从Azurea JCM-3275的基因组DNA克隆编码细胞色素P450的基因。我们克隆了一个完整的新P450基因（1209 bp），并将其命名为P450Um-1。其推导的氨基酸序列与CYP105亚家族的同源。进一步克隆可能含有天然启动子位点的上游区域，随后将具有上游区域的开放阅读框插入链霉菌高拷贝载体pIJ702中，得到表达质粒pNUm-1。 P450Um-1在青霉链霉菌TK24中特异性表达，该重组菌株将AS1387392转化为AS1429716，而没有任何氧化还原伴侣。这些结果表明，新型细菌P450 P450Um-1催化了AS1387392到AS1429716的羟基化。预期该所得重组菌株将是一种有效的生物催化剂，与此处测试的那些相比，可应用于更合适的氧化还原系统。

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