Multiple Oxidative Routes towards the Maturation of Nosiheptide
Multiple Oxidative Routes towards the Maturation of Nosiheptide
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那西肽成熟的多种氧化途径
DOI：
10.1002/cbic.201300427
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发表时间：
2013-09-02
期刊：
影响因子：
3.2
通讯作者：
Chen, Yijun
中科院分区：
文献类型：
--
作者：
Liu, Weiying;Xue, Yanjiu;Chen, Yijun
一个大环核、一个吲哚酸环和一个脱氢丙氨酸(DHA)尾巴。1的生物合成是通过核糖体合成和翻译后修饰肽(RIPP)系统进行的，在该系统中，至少有13个翻译后修饰发生，将13个残基的前体多肽转化为成熟的代谢物。序列分析和对基因功能的各种研究已经阐明了前体多肽的核糖体起源、吲哚酸环的形成和C末端尾部的成熟。根据1的生物合成基因簇，通过体内失活和定点突变，确定nosM为前体多肽基因。此外，由于基因失活后没有中间产物，nosDEFGHO负责骨架的形成，nosN和nosL与吲哚酸环的形成有关。此外，成熟的C-末端DHA尾部被NOSA官能化，通过烯胺脱烷基化作用于双-DHA中间体。虽然1的生物合成途径已经初步描绘出来，但涉及复杂修饰的详细步骤在很大程度上仍然难以捉摸。4b，5a]尽管氧化发生在大多数微生物次生代谢物的生物合成途径中，但不同的氧化酶及其各自底物的特殊性通常决定着生物合成的方向和过程。因此，1大环上两个羟基的存在促使我们在本研究中探索了氧化步骤及其与1成熟过程的关系。序列比对和系统发育分析表明，1的生物合成基因簇中的nosb和nosc很可能是细胞色素P450样单加氧酶(图S1)。进一步将编码蛋白NOSB和NOSC与其他已知的P450酶进行比较，发现它们有显著的相似性，包括一个保守的氨基酸序列、一个O2结合位点和一个C-末端的血红素结合结构域，该结构域具有与血红素配位的标志性半胱氨酸残基(图S2)。因此，随后研究了这两种可能的单加氧酶的功能作用，以深入了解它们在1成熟过程中的参与。首先，对NOSB进行基因敲除，以检测其对产物形成的影响。为了避免对下游基因NOSA表达的潜在影响，在诺西肽产生菌(野生型致密链霉菌ATCC 25421)中，采用框内缺失的方法使NOSB失活，从而产生突变菌株L1120(图1 A)。DnosB突变体L1120在对发酵提取物(图1B，泳道2)进行高效液相-紫外分析后，失去了产生1的能力，而产生了中间体2。中间体2随后从发酵液中分离纯化，用于结构测定。HR-ESI-MS分析表明m/z为1206.1596[M+H]为2(图S4A)，对应于分子式C51H43N13O11S6(m/z计算为：1206.1602)。此外，MS/MS和全套1D和2D-核磁共振数据证实了2的结构是Glu6上没有羟基的中间体(表S3，图S4，S5B和S6)，表明NOSB负责Glu6的g位羟基化。然后将携带pKL1120C的NOSB的单个副本引入L1120，以获得L1120C。得到的菌株恢复了1的生产(图1B，泳道6)，证明了NOSB在1的成熟过程中的必要性。为了验证NOSB的催化功能，我们在大肠杆菌BL21(DE3)中过表达了NOSB，并对重组NOSB进行了纯化，得到了均一的NOSB(图S15)。NOSB的紫外-可见光谱在418 nm处有一个最大吸收峰。加入Na2S2O4后，再用CO鼓泡，最大吸收波长移动到449 nm，显示出细胞色素P450样蛋白的特征(图S16)。氧化方案1.诺西肽(1)和中间体2-6的结构
a macrocyclic core, an indolic acid ring, and a dehydroalanine (Dha) tail. The biosynthesis of 1 was found to occur through a ribosomally synthesized and post-translationally modified peptide (RiPP) system, in which at least 13 post-translational modifications take place to convert a 13-residue precursor peptide into the mature metabolite. Sequence analyses and various investigations of gene functions have clarified the ribosomal origin of the precursor peptide, the formation of the indolic acid ring 4] and the maturation of the C-terminal tail. Based on the biosynthetic gene cluster of 1, nosM was confirmed to be the precursor peptide gene by in vivo inactivation and site-directed mutagenesis. In addition, nosDEFGHO were suggested to be responsible for framework formation because of the absence of intermediate production after gene inactivation, and nosN and nosL were identified to be associated with the formation of the indolic acid ring. Moreover, the mature C-terminal Dha tail was functionalized by NosA, which acts on the bis-Dha intermediate through the process of enamine dealkylation. Although a large picture of the biosynthetic pathway of 1 has been preliminarily drawn, the detailed steps involving complicated modifications remain largely elusive. 4b, 5a] Despite the fact that oxidation occurs in most biosynthetic pathways of microbial secondary metabolites, different oxidative enzymes and their respective substrate specificities usually determine the biosynthetic direction and process. Hence, the existence of two hydroxy groups on the macrocyclic moiety of 1 prompted us to exploit the oxidative steps and their relationship with the maturation process of 1 in the present study. Sequence alignments and phylogenetic analysis showed that nosB and nosC in the biosynthetic gene cluster of 1 are very likely cytochrome P450-like mono-oxygenases (Figure S1). Further comparison of the coding proteins NosB and NosC with other known P450 enzymes revealed remarkable similarity, including a conserved amino acid sequence, an O2 binding site, and a C-terminal heme-binding domain with the signature cysteine residue for the coordination of heme (Figure S2). Therefore, the functional roles of these two putative mono-oxygenases were subsequently investigated to gain insights into their involvements in the maturation of 1. First, gene knockout of nosB was carried out to examine the influence on product formation. To avoid potential effects on the expression of downstream gene nosA, in-frame deletion was employed to inactivate nosB in the nosiheptide-producing strain (wild-type S. actuosus ATCC 25421), leading to the generation of mutant strain L1120 (Figure 1 A). The DnosB mutant L1120, upon HPLC-UV analysis of the fermentation extracts (Figure 1 B, lane 2), lost the capability to produce 1, and intermediate 2 was generated instead. Intermediate 2 was subsequently isolated and purified from the culture broth for structural determination. HR-ESI-MS analysis showed m/z 1206.1596 [M+H] for 2 (Figure S4 A), corresponding to a molecular formula of C51H43N13O11S6 (m/z calcd: 1206.1602). Furthermore, MS/MS and full sets of 1Dand 2D-NMR data confirmed the structure of 2 to be an intermediate without the hydroxy group on Glu6 (Table S3, Figures S4, S5 B and S6), indicating that NosB is responsible for the hydroxylation of Glu6 at its g-position. A single copy of nosB carrying pKL1120C was then introduced to L1120 to give L1120C. The resulting strain restored production of 1 (Figure 1 B, lane 6), demonstrating the necessity of NosB in the maturation of 1. To verify the catalytic function of NosB, we overexpressed nosB in E. coli BL21(DE3), and the recombinant NosB was purified to homogeneity (Figure S15). UV–visible spectra of NosB exhibited a peak with maximum absorbance at 418 nm. After addition of Na2S2O4, followed by bubbling with CO, the maximum absorption was shifted to 449 nm, showing characteristics of cytochrome P450-like proteins (Figure S16). Oxidation Scheme 1. Structures of nosiheptide (1) and intermediates 2–6.