Engineering unnatural variants of plantazolicin through codon reprogramming.
Engineering unnatural variants of plantazolicin through codon reprogramming.
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DOI：
10.1021/cb4003392
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发表时间：
2013-09-20
影响因子：
4
通讯作者：
Mitchell, Douglas A.
中科院分区：
文献类型：
--
作者：
Deane, Caitlin D.;Melby, Joel O.;Molohon, Katie J.;Susarrey, Aziz R.;Mitchell, Douglas A.
Plantazolicin（PZN）是一种多杂环天然产物，其衍生自核糖体肽，对炭疽的病原体炭疽芽孢杆菌具有显著的抗生素选择性。为了同时建立PZN的结构-活性关系和生物合成途径的底物耐受性，大肠杆菌表达菌株被工程化以异源产生PZN类似物。通过定点诱变产生变异的PZN前体基因，然后通过质谱法筛选以评估翻译后修饰和通过E.杆菌从72个前体肽的筛选中，检测到29个PZN变体。这种类似物的收集提供了对翻译后修饰酶的选择性的深入了解，并建立了天然生物合成途径的边界。与其他研究的噻唑/恶唑修饰的微菌素不同，生物合成机制似乎对PZN的产生进行了微调，使得同源酶甚至不处理来自类似生物合成簇的其他天然存在的序列。修饰酶具有精细的选择性，仅在前体肽内预定义的位置安装杂环，而使相邻的残基未被修饰。几乎所有的取代位置通常窝藏杂环阻止成熟的PZN变体，虽然一些例外成功地产生缺乏杂环在倒数第二个残基。未检测到含有额外杂环的变体，尽管由于选择残基的不同氧化态，几种肽序列产生多个PZN变体。11个PZN变体以足够的量产生，以便于纯化和评估其抗菌活性，从而深入了解PZN的结构-活性关系。
Plantazolicin (PZN) is a polyheterocyclic natural product derived from a ribosomal peptide that harbors remarkable antibiotic selectivity for the causative agent of anthrax, Bacillus anthracis. To simultaneously establish the structure-activity relationship of PZN and the substrate tolerance of the biosynthetic pathway, an Escherichia coli expression strain was engineered to heterologously produce PZN analogs. Variant PZN precursor genes were produced by site-directed mutagenesis and later screened by mass spectrometry to assess posttranslational modification and export by E. coli. From a screen of 72 precursor peptides, 29 PZN variants were detected. This analog collection provided insight into the selectivity of the posttranslational modifying enzymes and established the boundaries of the natural biosynthetic pathway. Unlike other studied thiazole/oxazole-modified microcins, the biosynthetic machinery appeared to be finely tuned towards the production of PZN, such that the cognate enzymes did not process even other naturally occurring sequences from similar biosynthetic clusters. The modifying enzymes were exquisitely selective, installing heterocycles only at pre-defined positions within the precursor peptides while leaving neighboring residues unmodified. Nearly all substitutions at positions normally harboring heterocycles prevented maturation of a PZN variant, though some exceptions were successfully produced lacking a heterocycle at the penultimate residue. No variants containing additional heterocycles were detected, although several peptide sequences yielded multiple PZN variants as a result of varying oxidation states of select residues. Eleven PZN variants were produced in sufficient quantity to facilitate purification and assessment of their antibacterial activity, providing insight into the structure-activity relationship of PZN.
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影响因子：
15
作者：
Dunbar, Kyle L.;Mitchell, Douglas A.
通讯作者：
Mitchell, Douglas A.
影响因子：
15
作者：
KULIOPULOS, A;WALSH, CT
通讯作者：
WALSH, CT
DOI：
10.1126/science.1176667
发表时间：
2009-08-28
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Fischbach MA;Walsh CT
通讯作者：
Walsh CT
影响因子：
14.8
作者：
Dunbar, Kyle L.;Melby, Joel O.;Mitchell, Douglas A.
通讯作者：
Mitchell, Douglas A.
DOI：
10.1039/c1cc14281j
发表时间：
2012-01-14
期刊：
Chemical communications (Cambridge, England)
影响因子：
--
作者：
Li C;Zhang F;Kelly WL
通讯作者：
Kelly WL