Dowling Daniel P, Kung Yan, Croft Anna K, Taghizadeh Koli, Kelly Wendy L, Walsh Christopher T, Drennan Catherine L
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139;
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):12432-12437. doi: 10.1073/pnas.1608615113. Epub 2016 Oct 17.
Epothilones are thiazole-containing natural products with anticancer activity that are biosynthesized by polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) enzymes EpoA-F. A cyclization domain of EpoB (Cy) assembles the thiazole functionality from an acetyl group and l-cysteine via condensation, cyclization, and dehydration. The PKS carrier protein of EpoA contributes the acetyl moiety, guided by a docking domain, whereas an NRPS EpoB carrier protein contributes l-cysteine. To visualize the structure of a cyclization domain with an accompanying docking domain, we solved a 2.03-Å resolution structure of this bidomain EpoB unit, comprising residues M1-Q497 (62 kDa) of the 160-kDa EpoB protein. We find that the N-terminal docking domain is connected to the V-shaped Cy domain by a 20-residue linker but otherwise makes no contacts to Cy. Molecular dynamic simulations and additional crystal structures reveal a high degree of flexibility for this docking domain, emphasizing the modular nature of the components of PKS-NRPS hybrid systems. These structures further reveal two 20-Å-long channels that run from distant sites on the Cy domain to the active site at the core of the enzyme, allowing two carrier proteins to dock with Cy and deliver their substrates simultaneously. Through mutagenesis and activity assays, catalytic residues N335 and D449 have been identified. Surprisingly, these residues do not map to the location of the conserved HHxxxDG motif in the structurally homologous NRPS condensation (C) domain. Thus, although both C and Cy domains have the same basic fold, their active sites appear distinct.
埃坡霉素是一类含噻唑的具有抗癌活性的天然产物，由聚酮合酶（PKS）-非核糖体肽合成酶（NRPS）EpoA-F酶进行生物合成。EpoB的环化结构域（Cy）通过缩合、环化和脱水作用，从一个乙酰基和L-半胱氨酸组装出噻唑官能团。EpoA的PKS载体蛋白在一个对接结构域的引导下提供乙酰部分，而NRPS的EpoB载体蛋白提供L-半胱氨酸。为了可视化带有伴随对接结构域的环化结构域的结构，我们解析了这个双结构域EpoB单元的分辨率为2.03 Å的结构，其包含160 kDa的EpoB蛋白的M1-Q497残基（62 kDa）。我们发现N端对接结构域通过一个20个残基的连接子与V形的Cy结构域相连，但除此之外与Cy没有其他接触。分子动力学模拟和其他晶体结构显示这个对接结构域具有高度的灵活性，强调了PKS-NRPS杂合系统各组分的模块化性质。这些结构还进一步揭示了两条20 Å长的通道，它们从Cy结构域上的远处位点延伸到酶核心的活性位点，允许两个载体蛋白与Cy对接并同时递送它们的底物。通过诱变和活性测定，已鉴定出催化残基N335和D449。令人惊讶的是，这些残基并不位于结构同源的NRPS缩合（C）结构域中保守的HHxxxDG基序的位置。因此，尽管C结构域和Cy结构域具有相同的基本折叠，但它们的活性位点似乎不同。
