Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.
Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.
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DOI：
10.1021/acsbiomedchemau.2c00023
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发表时间：
2022-10-19
期刊：
影响因子：
--
通讯作者：
Peters, Reuben J.
中科院分区：
文献类型：
--
作者：
Lemke, Cody;Roach, Kristin;Ortega, Teresa;Tantillo, Dean J.;Siegel, Justin B.;Peters, Reuben J.
关键词：
毁灭性的人类病原体结核分枝杆菌（Mtb）能够寄生在肺泡巨噬细胞内的吞噬体区室，部分原因是其细胞表面脂质的活性。其中突出的是1-结核菌素-腺苷（1-TbAd），其是由Rv 3377 c编码的II类二萜环化酶产生的二萜类结核菌素（halima-5，13-二烯基）二磷酸的衍生物，在此称为MtHPS。考虑到已证明的1-TbAd作为Mtb毒力因子的能力以及Rv 3377 c对其生产的必要性，对MtHPS活性存在重大兴趣。II类二萜环化酶催化一般酸碱介导的碳阳离子级联反应，该反应由一般二萜类前体（E，E，E）-香叶基香叶基二磷酸中的末端烯烃的质子化引发，并由最终环化（有时也重排）中间体的去质子化终止。在这里，应用结构引导的诱变来表征有助于酶促酸活化的各种残基，以及鉴定MtHPS中的酶促碱基。特别是考虑到酶促碱基（Y 479 F）的保守取代通过早期未重排中间体的去质子化产生替代产物（labda-7，13-二烯基二磷酸）的能力，进行进一步的突变分析以引入潜在的替代催化碱基。将结果与机制分子建模相结合，以阐明这些突变如何影响这种重要酶的催化活性。这不仅提供了详细的结构-功能洞察MtHPS，但也进一步强调了惰性的MtHPS和II类二萜环化酶的活性位点更普遍的性质。
The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid–base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure–function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.
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