publications of the research group structural biology of
biosynthetic enzymes ([HIPS]SBBE)
http://hdl.handle.net/10033/620649
2024-03-28T09:13:43Z
2024-03-28T09:13:43Z
Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.
Sikandar, Asfandyar
Lopatniuk, Maria
Luzhetskyy, Andriy
Müller, Rolf
Koehnke, Jesko
http://hdl.handle.net/10033/623172
2022-04-13T02:01:25Z
2022-03-09T00:00:00Z
Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway.
Sikandar, Asfandyar; Lopatniuk, Maria; Luzhetskyy, Andriy; Müller, Rolf; Koehnke, Jesko
Thioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.
Thioholgamides are ribosomally synthesized and posttranslationally modified peptides (RiPPs), with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes─ThoH/I and ThoC/D. Harnessing the power of AlphaFold, we deduce that ThoD acts as a lyase and also proposes putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys, and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation─a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide. © 2022 American Chemical Society. All rights reserved.
2022-03-09T00:00:00Z
Characterization of the Stereoselective P450 Enzyme BotCYP Enables the Biosynthesis of the Bottromycin Core Scaffold.
Adam, Sebastian
Franz, Laura
Milhim, Mohammed
Bernhardt, Rita
Kalinina, Olga V
Koehnke, Jesko
http://hdl.handle.net/10033/622954
2021-07-24T01:51:17Z
2020-11-28T00:00:00Z
Characterization of the Stereoselective P450 Enzyme BotCYP Enables the Biosynthesis of the Bottromycin Core Scaffold.
Adam, Sebastian; Franz, Laura; Milhim, Mohammed; Bernhardt, Rita; Kalinina, Olga V; Koehnke, Jesko
Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.
2020-11-28T00:00:00Z
Human IFITM3 restricts chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction.
Franz, Sergej
Pott, Fabian
Zillinger, Thomas
Schüler, Christiane
Dapa, Sandra
Fischer, Carlo
Passos, Vânia
Stenzel, Saskia
Chen, Fangfang
Döhner, Katinka
Hartmann, Gunther
Sodeik, Beate
Pessler, Frank
Simmons, Graham
Drexler, Jan Felix
Goffinet, Christine
http://hdl.handle.net/10033/622918
2021-07-02T01:42:23Z
2021-06-02T00:00:00Z
Human IFITM3 restricts chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction.
Franz, Sergej; Pott, Fabian; Zillinger, Thomas; Schüler, Christiane; Dapa, Sandra; Fischer, Carlo; Passos, Vânia; Stenzel, Saskia; Chen, Fangfang; Döhner, Katinka; Hartmann, Gunther; Sodeik, Beate; Pessler, Frank; Simmons, Graham; Drexler, Jan Felix; Goffinet, Christine
Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3 Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.
2021-06-02T00:00:00Z
Structure-Activity Relationship and Mode-of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.
Walter, Isabell
Adam, Sebastian
Gentilini, Maria Virginia
Kany, Andreas M
Brengel, Christian
Thomann, Andreas
Sparwasser, Tim
Köhnke, Jesko
Hartmann, Rolf W
http://hdl.handle.net/10033/622915
2021-07-02T01:42:46Z
2021-05-19T00:00:00Z
Structure-Activity Relationship and Mode-of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.
Walter, Isabell; Adam, Sebastian; Gentilini, Maria Virginia; Kany, Andreas M; Brengel, Christian; Thomann, Andreas; Sparwasser, Tim; Köhnke, Jesko; Hartmann, Rolf W
CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.
2021-05-19T00:00:00Z