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

【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 DH_2)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 DH_2的底物类似物waq-1,建立和优化了Ansa DH_2的体外生化反应体系;并利用ESI-MS~2的方法鉴定了Ansa DH_2催化形成的终产物的结构,确定了Ansa DH_2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 DH_2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 DH_2和Ansa DH_3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 DH_2底物类似物waq-1,终产率为17.81%;建立并优化了Ansa DH_2的生化反应条件,使36 h的底物转化率达到45%;通过对Ansa DH_2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 DH_2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 DH_2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 DH_2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。

[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 theα-βdehydration 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.Theα-βdehydration 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 itsα-βdehydration activity,but also shed light on further mechanistic study of olefin shift.