A Ribonucleotide Reductase-Like Electron Transfer System in the Nitroaryl-Forming N-Oxygenase AurF
A Ribonucleotide Reductase-Like Electron Transfer System in the Nitroaryl-Forming N-Oxygenase AurF
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DOI：
10.1002/cbic.201100138
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发表时间：
2011-08-16
期刊：
影响因子：
3.2
通讯作者：
Hertweck, Christian
中科院分区：
文献类型：
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作者：
Fries, Alexander;Bretschneider, Tom;Hertweck, Christian
许多天然产物具有重要的生物活性，如抗菌、抗真菌、杀虫、抗肿瘤等。最著名的代表可能是抗生素氯霉素、抗真菌剂硝吡咯和抗增殖的聚酮化合物金霉素（1）。两个主要的生物合成途径，硝化和氨基的N-氧化，导致天然硝基化合物。[1]令人惊讶的是，到目前为止，只有两种真正的芳胺N-加氧酶被鉴定出来：来自荧光假单胞菌中的吡咯硝基蛋白途径的PrnD，[2]和在硫黄链霉菌中产生金硫素生物合成的对硝基苯甲酸酯（PNBA）构件的AurF。[3]由于其显著的化学和区域选择性，AurF作为生物催化剂在技术应用中具有很高的潜力。[4，5]虽然这种不寻常的生物催化剂是前所未有的，但最近通过基因组分析在未知的生物合成聚酮合酶（PKS）和非核糖体肽合酶（NRPS）基因簇中鉴定了auRF的同源物。[6]通过大量的体内和体外研究，我们可以表明，氨基底物的N-氧化是逐步发生的，涉及羟胺和亚硝基中间体。[7]此外，我们可以证明锰参与AuRF的催化作用，并能够提出第一个N-加氧酶的X射线结构。[8，9]后来，另一个小组报告了AuRF的含铁变体的晶体结构。[10]在这两种结构中，整体链折叠是相似的，并在活性中心具有双核金属簇。铁和锰变体都表现出体内和体外活性，这导致了对AURF的天然形式的一些争论，也考虑了混合的Fe/Mn核。[11然而，对于两种结构相关的双加氧酶，已经证明氧活化可以通过任一种金属实现。[13]非常引人注目的是，金硫蛋白生物合成基因簇不包含任何编码电子转移链组分的基因，所述电子转移链组分通常由铁氧还蛋白、铁氧还蛋白还原酶和NAD（P）H组成。[14]然而，AurF已被证明在至少三种不同的生物体中具有体内活性：在其天然宿主硫黄链霉菌[15]以及异源宿主变铅青链霉菌[7]和E.杆菌[4]此外，纯化的酶可以通过使用过氧化氢分流器在体外再生。[4]尽管天然相互作用配偶体仍然未知，但可以通过使用来自鱼腥藻属PCC 7119的替代铁氧还蛋白和铁氧还蛋白还原酶（Δ9去饱和酶）在体外重建AURF。[10]然而，到目前为止，电子如何穿梭到活性部位的金属上仍然是难以捉摸的。在这里，我们提供了对这种不寻常的硝基形成酶的电子传递系统的第一个见解（方案1）。尽管AurF与已知酶没有明显的序列同源性，但通过DALI分析[16]，我们发现链折叠和生物金属簇与核糖核苷酸还原酶（RNR），甲烷单加氧酶/羟化酶和酰基去饱和酶/脂肪酸还原酶非常匹配。在所有情况下，金属簇嵌入在一个大的螺旋束和协调的两个（D/E）EXXH基序。此外，con-
Many natural products endowed with the rare nitro moiety exhibit important biological activities such as antibiotic, antifungal, insecticidal, or antitumoral. The perhaps best-known representatives are the antibiotic chloramphenicol, the antifungal agent pyrrolnitrin, and the antiproliferative polyketide aureothin (1). Two principal biosynthetic pathways, nitration and N-oxygenation of amino groups, lead to natural nitro compounds.[1] Surprisingly, up to now only two genuine arylamine N-oxygenases have been identified: PrnD from the pyrrolnitrin pathway in Pseudomonas fluorescens,[2] and AurF, which generates the p-nitrobenzoate (PNBA) building block for aureothin biosynthesis in Streptomyces thioluteus.[3] Due to its remarkable chemo-and regioselectivity, AurF has a high potential as biocatalyst for technical applications.[4, 5] Although this unusual biocatalyst has been unprecedented, recently homologues of aurF have been identified through genomic analyses in yetunknown biosynthetic polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) gene clusters.[6] Through a number of in vivo and in vitro studies, we could show that N-oxygenation of the amino substrates occurs stepwise and involves hydroxylamine and nitroso intermediates.[7] Furthermore, we could prove the participation of manganese in the catalytic action of AurF and were able to present the first X-ray structure of an N-oxygenase.[8, 9] Later, another group reported the crystal structure of an iron-containing variant of AurF.[10] In both structures, the overall chain folds were similar and featured a binuclear metal cluster in the active center. Both iron and manganese variants exhibit in vivo and in vitro activity, which led to some debate on the native form of AurF, also considering mixed Fe/Mn nuclei.[11, 12] Nevertheless, it has been demonstrated for two structurally related dioxygenases that oxygen activation can be achieved by either metal.[13] What is quite striking is that the aureothin biosynthesis gene cluster does not contain any gene coding for components of an electron transfer chain, which usually consists of a ferredoxin, a ferredoxin reductase and NAD (P) H.[14] However, AurF has been proven to be active in vivo in at least three different organisms: in its natural host Streptomyces thioluteus,[15] as well as in the heterologous hosts Streptomyces lividans [7] and E. coli.[4] Moreover, the purified enzyme can be regenerated in vitro by using the peroxide shunt.[4] Whereas the native interaction partners are still unknown, AurF could be reconstituted in vitro by using surrogate ferredoxin and ferredoxin reductase from Anabaena sp. PCC 7119 (Δ9 desaturase).[10] However, to date it has remained elusive how electrons are shuttled to the metals in the active site. Here we provide the first insight into the electron transport system of this unusual nitro-group-forming enzyme (Scheme 1).Although there is no obvious sequence homology of AurF with known enzymes, by DALI analyses [16] we found that the chain fold and biometal cluster match remarkably well with ribonucleotide reductases (RNR), methane monooxygenases/hydroxylases, and acyl desaturases/fatty acid reductases. In all cases, the metal cluster is embedded in a large helix bundle and coordinated by two (D/E) EXXH motifs. Furthermore, con-