Characterization of the Stereoselective P450 Enzyme BotCYP Enables the <i>In Vitro</i> Biosynthesis of the Bottromycin Core Scaffold

Adam, Sebastian; Franz, Laura; Milhim, Mohammed; Bernhardt, Rita; Kalinina, Olga; Koehnke, Jesko

doi:10.1021/jacs.0c10361

Cited by 9 publications

(17 citation statements)

References 31 publications

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“…The reaction mixture containing both epimerase BotH and P450 BotCYP led to the reaction to completion, rather than containing only BotCYP. The epimerase enzyme BotH first epimerizes the structures obtained by several enzymatic reactions, and then, BotCYP preferentially oxidatively decarboxylated the epimer containing d ‐aspartic acid (Adam et al, 2020). TbtJ1 and TbtJ2 could perform selective cyclopropanation of dehydroalanine residues within complex linear and cyclic RiPP cores in thiomuracin biosynthesis, which is another case of unusual reaction facilitated by P450 (Gober et al, 2017).…”

Section: Structure and Function Of P450s Involved In Antibiotics Bios...mentioning

confidence: 99%

Biocatalytic role of cytochrome P450s to produce antibiotics: A review

Adhikari,

Shakya,

Shrestha

et al. 2023

Biotech & Bioengineering

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Cytochrome P450s belong to a family of heme‐binding monooxygenases, which catalyze regio‐ and stereospecific functionalisation of C–H, C–C, and C–N bonds, including heteroatom oxidation, oxidative C–C bond cleavages, and nitrene transfer. P450s are considered useful biocatalysts for the production of pharmaceutical products, fine chemicals, and bioremediating agents. Despite having tremendous biotechnological potential, being heme‐monooxygenases, P450s require either autologous or heterologous redox partner(s) to perform chemical transformations. Randomly distributed P450s throughout a bacterial genome and devoid of particular redox partners in natural products biosynthetic gene clusters (BGCs) showed an extra challenge to reveal their pharmaceutical potential. However, continuous efforts have been made to understand their involvement in antibiotic biosynthesis and their modification, and this review focused on such BGCs. Here, particularly, we have discussed the role of P450s involved in the production of macrolides and aminocoumarin antibiotics, nonribosomal peptide (NRPSs) antibiotics, ribosomally synthesized and post‐translationally modified peptide (RiPPs) antibiotics, and others. Several reactions catalyzed by P450s, as well as the role of their redox partners involved in the BGCs of various antibiotics and their derivatives, have been primarily addressed in this review, which would be useful in further exploration of P450s for the biosynthesis of new therapeutics.

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Section: Structure and Function Of P450s Involved In Antibiotics Bios...mentioning

confidence: 99%

Biocatalytic role of cytochrome P450s to produce antibiotics: A review

Adhikari,

Shakya,

Shrestha

et al. 2023

Biotech & Bioengineering

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“…30 Recently, BotCYP was reconstituted using three enzymes: BotCYP, BmCPR, and Fdx2 (from Bacillus megaterium 5 ) with substrate a/b (a = macroamidated, thiozoline product, b = epimerase, BotH-treated com-pound a) in the presence of NADPH resulting in a decarboxylated and oxidized reaction product with very poor yield (<25%). 28 To find a reason for low yield, equimolar mixture of L-Asp and D-Asp was used as a substrate and the product mixture was analyzed by LC-MS. Only D-Asp-containing substrate showed oxidative decarboxylation indicating that BotCYP preferentially accepts the D-isomer as a substrate. This confirmed that epimerization of L-Asp to D-Asp by epimerase BotH proceeds the BotCYP reaction.…”

Section: Oxidative Decarboxylation (Botcyp)mentioning

confidence: 99%

“…It indicates that during oxidative decarboxylation to the aromatic thiazole, the configuration of the C α -position of Asp7 was retained. 28…”

Section: Enzymology Of Bottromycin Biosynthesismentioning

confidence: 99%

“…Bottromycin BGC was discovered in 2012 establishing that it is a ribosomally synthesized peptide, [14][15][16]23 however, in vitro biochemical studies on various enzymes on the pathway have not been reported until recently. [24][25][26][27][28][29] The BGC from S. sp. BC16019 is shown in Figure 2 with annotated functions of the enzymes.…”

Section: Cluster Account Synlettmentioning

confidence: 99%

“…This resulted in a peak with a similar LC retention time and MS/MS fragmentation patterns as the previous oxidative decarboxylated It indicates that during oxidative decarboxylation to the aromatic thiazole, the configuration of the C  -position of Asp7 was retained. 28 A mechanism for this unique cytochrome P450 enzyme mediated oxidative decarboxylation is proposed in Scheme 7. Abstraction of -hydrogen from the thiazoline intermediate 30 by the heme Fe (IV)-oxo cation radical intermediate 31 results in the substrate radical 32 and heme Fe (IV)-oxo intermediate 33.…”

Section: Oxidative Decarboxylation (Botcyp)mentioning

confidence: 99%

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Recent Advancements in Bottromycin Biosynthesis

Mahanta¹,

PH²,

KS³

et al. 2022

Synlett

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Bottromycin is a structurally complex cyclic peptidic compound isolated from Streptomyces bottropensis and related organisms and belongs to the RiPP family of natural products (ribosomally synthesized and post-translationally modified peptides). It exhibits potent antibacterial properties against gram-positive pathogens (including drug resistant strains such as MRSA, MIC 1 μg/mL and VRE, MIC 0.5 μg/mL) and mycoplasma. Bottromycin blocks the binding of the aminoacyl-tRNA to the A-site on the 50S ribosome and hence inhibits protein synthesis. Bottromycins contain structurally diverse post-translational modifications (PTMs) on a small peptide (GPVVVFDC) including a unique macrocyclic amidine, rare β-methylation, terminal thiazole heterocycle, oxidative decarboxylation, and Asp epimerization, among others. It exhibits a precursor peptide organization with a C-terminal follower peptide and a N-terminal core peptide. There are several new studies reported recently which gave detailed insights into the bottromycin biosynthesis pathway. This Account highlights the current advancements in understanding the biosynthetic pathway of bottromycin focusing mainly on the biochemically and structurally characterized enzymes and intricate details of the peptide–protein biophysical interactions. These studies have provided a strong foundation for conducting combinatorial biosynthesis and synthetic biological studies to create novel bottromycin variants for therapeutic applications.1 Introduction2 Biosynthetic Pathway for Bottromycin3 Enzymology of Bottromycin Biosynthesis3.1 Cleavage of Methionine (BotP)3.2 Radical SAM Methyltransferases (BotRMT1, BotRMT2, BotRMT3)3.3 ATP-Dependent YcaO Enzymes3.3.1 Thiazoline Formation by BotC3.3.2 Macrolactamidine Formation by BotCD3.4 Follower Peptide Hydrolysis (BotAH)3.5 Aspartate Epimerization (BotH)3.6 Oxidative Decarboxylation (BotCYP)3.7 O-Methyltransferase (BotOMT)4 Heterologous Bottromycin Production and Analogue Preparation5 Summary and Outlook

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Cytochromes P450 Associated with the Biosyntheses of Ribosomally Synthesized and Post-translationally Modified Peptides

Zhong

2023

ACS Bio Med Chem Au

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of exponentially increased natural products with characteristic chemical structures, topologies, and biosynthetic mechanisms as well as exceptional bioactivities including antibacteria, antitumors, and antiviruses. The biosynthesis of RiPP proceeds via a ribosomally assembled precursor peptide that undergoes varied post-translational modifications to generate a mature peptide. Cytochrome P450 (CYP or P450) monooxygenases are a superfamily of heme-containing enzymes that span a wide range of secondary metabolite biosynthetic pathways due to their broad substrate scopes and excellent catalytic versatility. In contrast to the enormous quantities of RiPPs and P450s, the P450 associated RiPP biosynthesis is comparatively limited, with most of their functions and timings remaining mysterious. Herein, this Review aims to provide an overview on the striking roles of P450s in RiPP biosyntheses uncovered to date and to illustrate their remarkable functions, mechanisms, as well as remaining challenges. This will shed light on novel P450 discovery and characterizations in RiPP biosyntheses.

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Characterization of the Stereoselective P450 Enzyme BotCYP Enables the In Vitro Biosynthesis of the Bottromycin Core Scaffold

Abstract: Bottromycins are ribosomally synthesized and posttranslationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillinresistant Staphylococcus aureus. The total synthesis of bottromycins

Cited by 9 publications

References 31 publications

Biocatalytic role of cytochrome P450s to produce antibiotics: A review

Biocatalytic role of cytochrome P450s to produce antibiotics: A review

Recent Advancements in Bottromycin Biosynthesis

Cytochromes P450 Associated with the Biosyntheses of Ribosomally Synthesized and Post-translationally Modified Peptides

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