Modular Enzymatic Assembly Lines for Antibiotics

描述（由申请人提供）：Nature在多模块酶组装线上组装数千种聚酮（PK）和非核糖体肽（NRP）天然产物抗生素。生长链作为一系列延长的共价酰基-S-蛋白中间体被拴系，通过磷酸泛酰巯基乙胺基链连接到载体蛋白结构域，通常每个模块一个。该建议探讨了抗生素天然产物中两组成熟步骤的催化机制和化学逻辑。新生支架通过专用酶进行定制反应，这些酶通常对靶生物活性的成熟至关重要。在具体目标1中，我们检查了NRP和NR-PK杂合体的两种类型的氧化剪裁酶的范围和机制：卤素酶和加氧酶。定制卤化酶使用FADH 2或单核非血红素Fe II，这取决于抗生素中碳位点的电子需求。我们计划使用卤化酶基因探针克隆生物合成基因簇，例如库茨内莱德，并检查卤化剪裁的范围。我们还将研究在抗生素支架成熟中起作用的定制加氧酶的活性和机制。具体目标2解决了在链延长和终止步骤期间涉及新型化学的组装线中的催化结构域。这些包括取代酰胺的酯骨架键，作为反式作用的缩合结构域的转氨酶同系物的募集，C-末端吡咯烷二酮环的形成，以及还原性链终止步骤。

DESCRIPTION (provided by applicant):Nature assembles thousands of polyketide (PK) and nonribosomal peptide (NRP) natural product antibiotics on multimodular enzymatic assembly lines. Growing chains are tethered as a series of elongating covalent acyl-S-protein intermediates, attached by phosphopantetheinyl chains to carrier protein domains, typically one per module. This proposal examines catalytic machinery and chemical logic for two sets of maturation steps in antibiotic natural products. Nascent scaffolds are subject to tailoring reactions by dedicated enzymes that often are crucial for maturation of target biological activities. In Specific Aim 1 we prose examination of scope and mechanism of two types of oxidative tailoring enzymes for NRP and NR-PK hybrids: halogenases and oxygenases. Tailoring halogenases use either FADH2 or mononuclear nonheme FeII, dependent on electronic demand of the carbon site in the antibiotic. We plan to use halogenase gene probes to clone biosynthetic gene clusters, e.g. for kutzneride, and examine scope of the halogenative tailoring. We shall also examine the activity and mechanism of tailoring oxygenases that act in antibiotic scaffold maturations. Specific Aim 2 addresses catalytic domains in assembly lines involved in novel chemistry during chain elongation and termination steps. These include ester backbone linkages in place of amides, the recruitment of transglutaminase homologs as condensation domains that act in trans, formation of C-terminal pyrrolidinedione rings, and reductive chain termination steps.