安丝菌素聚酮合酶模块2中脱水酶结构域的生化功能

Biochemical analysis of the dehydratase domain in module 2 involved in ansamitocin biosynthesis

摘要:

[目的] I型聚酮合酶（Polyketide synthase，PKS）模块中不同的修饰是聚酮类化合物结构多样性的重要原因之一。抗癌药物安丝菌素化学结构中C11-C14区域存在特殊的双键迁移结构，可能与聚酮合酶模块2或者3中脱水酶结构域（Dehydratase，DH）的催化密切相关，本研究通过探究聚酮合酶模块2中DH结构域（Ansa DH₂）的生化功能，确定其在安丝菌素聚酮链延伸过程中的脱水功能。[方法] 本文以安丝菌素产生菌Actinosynnema pretiosum subsp. pretiosum ATCC 31280为研究材料，首先，对安丝菌素聚酮合酶模块中的4个不同DH结构域进行了生物信息学分析；然后，利用化学方法合成了Ansa DH₂的底物类似物waq-1，建立和优化了Ansa DH₂的体外生化反应体系；并利用ESI-MS²的方法鉴定了Ansa DH₂催化形成的终产物的结构，确定了Ansa DH₂在安丝菌素聚酮链延伸中的α-β脱水作用；最后，通过定点突变的策略，对Ansa DH₂的关键氨基酸进行了鉴定。[结果] 通过生物信息学分析显示Ansa DH₂和Ansa DH₃与已报道的催化双键迁移的DH结构域进化关系近；通过六步线性化学反应合成了Ansa DH₂底物类似物waq-1，终产率为17.81%；建立并优化了Ansa DH₂的生化反应条件，使36 h的底物转化率达到45%；通过对Ansa DH₂催化形成的终产物的结构鉴定，证明安丝菌素聚酮链延伸过程中Ansa DH₂催化底物类似物发生了α-β脱水；最后，氨基酸的定点突变证明了Ansa DH₂第48位的组氨酸（H）是脱水活性的必需氨基酸。[结论] 本研究证明了Ansa DH₂具有催化α-β脱水的功能，丰富了DH结构域的研究，为后续研究安丝菌素聚酮链延伸过程中的双键迁移奠定了基础。

Abstract:

[Objective] Multifarious modular modifications in type I polyketide synthase (PKS) serve as crucial contributing factors for the diversity of polyketides. Ansamitocins, an antitumor agent, possess unique olefin shifts in the region of C11-C14, which might be catalyzed by dehydratase domain in PKS module 2 and 3. We evaluated the biochemical function of dehydratase domain in module 2 (Ansa DH₂). [Methods] Using ansamitocin-producing strain Actinosynnema pretiosum subsp. pretiosum ATCC 31280, we chose four different Ansa DHs in the ansamitocin biosynthetic modules to achieve bioinformatics analysis. Coupled with chemical synthesis of an analogue substrate waq-1 of Ansa DH₂, we presented our in vitro investigations. ESI-MS² analysis revealed the structure of the final product, which confirmed the α-β dehydration activity of Ansa DH₂. Ultimately, the correlation between the catalytic function and requisite amino acid residues has been determined through site-directed mutagenesis. [Results] Bioinformatics analysis suggests that Ansa DH₂ and Ansa DH₃ were closely related to those known DHs, which are responsible for olefin shift. An effective synthetic route was accomplished to afford waq-1 in 17.81% yield over six linear steps. The conversion rate of waq-1 reached 45% after 36 h by optimized in vitro enzymatic reactions. The α-β dehydration activity of Ansa DH₂ was reaffirmed by fragmentations in MS² profiles. Residue His48 in Ansa DH₂ was turned out to be indispensable for dehydration function by site-directed mutagenesis. [Conclusion] This work focused on the biochemical function of Ansa DH₂, which not only verified its α-β dehydration activity, but also shed light on further mechanistic study of olefin shift.