Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

Zhu, Yiguang; Zhang, Qingbo; Fang, Chunyan; Zhang, Yingli; Ma, Liang; Liu, Zhiwen; Zhai, Shilan; Peng, Jing; Zhu, Weiming

doi:10.1002/ange.202004978

Cited by 5 publications

(4 citation statements)

References 44 publications

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“…Thus, our DFT results indicate the direct hydroxylation in the C2 position is less likely and a preceding epoxide intermediate could be needed. In addition to the FPMOs, two cytochrome P450 monooxygenases, Aspergillusderived FtmG 37 and actinomycete-derived CyaH 54 , could also catalyze the formation of spirooxindole through a proposed radical-mediated hydroxylation and semipinacol rearrangement.…”

Section: Discussionmentioning

confidence: 99%

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Liu

Zhao

et al. 2021

Nat Commun

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Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

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Section: Discussionmentioning

confidence: 99%

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Liu

Zhao

et al. 2021

Nat Commun

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“…Our data shows that ferredoxin provides optimal coupling of CYP2A_B49 to photosynthetic reducing power. Although coupling P450 activity to photosynthetic reducing power is still a relatively niche field, plant and cyanobacterial ferredoxins has been used as surrogate electron transfer partners in many reconstituted P450 systems ( Wirtz et al., 2000 ; Jackson et al., 2002 ; Goñi et al., 2009 ; Jensen et al., 2012 ; Zhang et al., 2018 ; Mie et al., 2020 ; Zhu et al., 2020 ; Liu et al., 2022 ). Recently a survey of 16 microbial ferredoxins found ferredoxin from the cyanobacteria Synechococcus elongatus PCC 7942 to be the most efficient redox partner for 5 different P450s ( Zhang et al., 2018 ).…”

Section: Discussionmentioning

confidence: 99%

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts

et al. 2023

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Photosynthetic organelles offer attractive features for engineering small molecule bioproduction by their ability to convert solar energy into chemical energy required for metabolism. The possibility to couple biochemical production directly to photosynthetic assimilation as a source of energy and substrates has intrigued metabolic engineers. Specifically, the chemical diversity found in plants often relies on cytochrome P450-mediated hydroxylations that depend on reductant supply for catalysis and which often lead to metabolic bottlenecks for heterologous production of complex molecules. By directing P450 enzymes to plant chloroplasts one can elegantly deal with such redox prerequisites. In this study, we explore the capacity of the plant photosynthetic machinery to drive P450-dependent formation of the indigo precursor indoxyl-β-D-glucoside (indican) by targeting an engineered indican biosynthetic pathway to tobacco (Nicotiana benthamiana) chloroplasts. We show that both native and engineered variants belonging to the human CYP2 family are catalytically active in chloroplasts when driven by photosynthetic reducing power and optimize construct designs to improve productivity. However, while increasing supply of tryptophan leads to an increase in indole accumulation, it does not improve indican productivity, suggesting that P450 activity limits overall productivity. Co-expression of different redox partners also does not improve productivity, indicating that supply of reducing power is not a bottleneck. Finally, in vitro kinetic measurements showed that the different redox partners were efficiently reduced by photosystem I but plant ferredoxin provided the highest light-dependent P450 activity. This study demonstrates the inherent ability of photosynthesis to support P450-dependent metabolic pathways. Plants and photosynthetic microbes are therefore uniquely suited for engineering P450-dependent metabolic pathways regardless of enzyme origin. Our findings have implications for metabolic engineering in photosynthetic hosts for production of high-value chemicals or drug metabolites for pharmacological studies.

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“…Importantly, Zhang et al discovered that cyanogramide D ( 1 ) serves as a biosynthetic precursor of the spirooxindole alkaloid cyanogramide ( 2 , Figure 1 ). 1 Cyanogramide ( 2 ) is able to restore the cytotoxicity of adriamycin and vincristine against the resistant cell lines K562/A02, MCF-7/Adr and KB/VCR, presumably by inhibition of the P-glycoprotein, an ATP-dependent drug efflux pump. 3…”

mentioning

confidence: 99%

“…Cyanogramides B, C, D ( 1 , Figure 1 ), and marinacarbolines K ( 3 ) and L ( 4 , Scheme 1 ) share a characteristic 2-methoxy-2-methylimidazolidinone ring, which is anellated at the pyridone ring, resulting in a tetracyclic skeleton. 1 2 Cytotoxicities at single-digit micromolar concentrations against the human glioma cell line U87MG have been observed for cyanogramide D ( 1 ) and cyanogramide C ( 5 , Scheme 1 ). Importantly, Zhang et al discovered that cyanogramide D ( 1 ) serves as a biosynthetic precursor of the spirooxindole alkaloid cyanogramide ( 2 , Figure 1 ).…”

mentioning

confidence: 99%

N-Oxide Route to the Marine Natural Product Cyanogramide D

Lindel,

Sarnes,

Struck

et al. 2023

Synthesis

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The first synthesis of the marine natural product cyanogramide D is reported. The key step is the acetylation of a β-carboline N-oxide, followed by acetyl migration. Since in this particular case it was not possible to incorporate the styryl side chain by Buchwald coupling, a phenylethanolamine side chain was attached, which was dehydrated with Martin's sulfurane after assembly of the tetracycle. Pentacyclic products were obtained under Appel conditions. The synthesis will facilitate the exploration of the biomimetic oxidative spirocyclization of cyanogramide D to the spirooxindole cyanogramide.

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Refactoring the Concise Biosynthetic Pathway of Cyanogramide Unveils Spirooxindole Formation Catalyzed by a P450 Enzyme

Cited by 5 publications

References 44 publications

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Exploiting photosynthesis-driven P450 activity to produce indican in tobacco chloroplasts

N-Oxide Route to the Marine Natural Product Cyanogramide D

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