Structure and Function of NzeB, a Versatile C-C and C-N Bond-Forming Diketopiperazine Dimerase.
Structure and Function of NzeB, a Versatile C-C and C-N Bond-Forming Diketopiperazine Dimerase.
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一种多功能的C-C和C-N键形成二酮基哌嗪二聚体NzeB的结构和功能。
DOI：
10.1021/jacs.0c06312
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发表时间：
2020-10-14
影响因子：
15
通讯作者：
Sherman DH
中科院分区：
文献类型：
--
作者：
Shende VV;Khatri Y;Newmister SA;Sanders JN;Lindovska P;Yu F;Doyon TJ;Kim J;Houk KN;Movassaghi M;Sherman DH
二聚二酮哌嗪（DKPs）生物碱是一个多样化的天然产物（NPs）家族，其独特的结构架构和生物活性激发了开发新的合成方法以获得这些分子。然而，缺乏能够从单一单体选择性形成结构和立体异构二聚体的催化剂控制的方法。为了解决这一长期存在的合成挑战，我们试图表征组装这些NP用于生物催化合成的生物合成酶。基因组挖掘使得能够鉴定细胞色素P450 NzeB（来源于链霉菌NRRL F-5053），其催化分子间碳-碳（C-C）和碳-氮（C-N）键的形成，产生从细菌来源分离的所有目前已知的DKP二聚体支架。为了确定灵活的网站，立体，和NzeB的化学选择性的分子基础，我们获得了高分辨率的晶体结构（1.5nm）的蛋白质与天然和非天然底物的复合物。据我们所知，这是催化直接分子间C-H胺化的氧化酶的第一个晶体结构。采用定点诱变来评估单个活性位点残基在引导选择性DKP二聚化中发挥的作用。最后，采用计算方法来评估关于NzeB功能及其催化C-C和C-N键形成的能力的可能机制。这些结果为NzeB的催化多功能性提供了结构和计算依据，并为控制CYP 450二酮哌嗪二聚酶选择性的变量提供了新的见解。
The dimeric diketopiperazine (DKPs) alkaloids are a diverse family of natural products (NPs) whose unique structural architectures and biological activities have inspired the development of new synthetic methodology to access these molecules. However, catalyst-controlled methods that enable the selective formation of constitutional and stereoisomeric dimers from a single monomer are lacking. To resolve this long-standing synthetic challenge, we sought to characterize the biosynthetic enzymes that assemble these NPs for application in biocatalytic syntheses. Genome mining enabled identification of the cytochrome P450, NzeB (derived from Streptomyces sp. NRRL F-5053), which catalyzes both intermolecular carbon-carbon (C–C) and carbon-nitrogen (C–N) bond formation, generating all currently known DKP dimer scaffolds isolated from bacterial sources. To identify the molecular basis for the flexible site-, stereo-, and chemoselectivity of NzeB, we obtained high-resolution crystal structures (1.5Å) of the protein in complex with native and non-native substrates. This, to our knowledge, represents the first crystal structure of an oxidase catalyzing direct, intermolecular C–H amination. Site-directed mutagenesis was employed to assess the role individual active site residues play in guiding selective DKP dimerization. Finally, computational approaches were employed to evaluate plausible mechanisms regarding NzeB function and its ability to catalyze both C–C and C–N bond formation. These results provide a structural and computational rationale for the catalytic versatility of NzeB, as well as new insights into variables that control selectivity of CYP450 diketopiperazine dimerases.
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影响因子：
18.3
作者：
Kim J;Movassaghi M
通讯作者：
Movassaghi M
DOI：
10.1016/j.bbapap.2015.04.015
发表时间：
2015-08
影响因子：
3.2
作者：
Gerlt, John A.;Bouvier, Jason T.;Davidson, Daniel B.;Imker, Heidi J.;Sadkhin, Boris;Slater, David R.;Whalen, Katie L.
通讯作者：
Whalen, Katie L.
影响因子：
2.9
作者：
Giessen, Tobias W.;von Tesmar, Alexander M.;Marahiel, Mohamed A.
通讯作者：
Marahiel, Mohamed A.
影响因子：
15
作者：
GEBLER, JC;WOODSIDE, AB;POULTER, CD
通讯作者：
POULTER, CD
影响因子：
16.6
作者：
Liu, Jing;Xie, Xiulan;Li, Shu-Ming
通讯作者：
Li, Shu-Ming