Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

Sikandar, Asfandyar; Lopatniuk, Maria; Luzhetskyy, Andriy; Müller, Rolf; Koehnke, Jesko

doi:10.1021/jacs.2c00402

Cited by 23 publications

(36 citation statements)

References 49 publications

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“…Furthermore, TvaC S‑87 was also identified as sharing similarity to the kinase domain of the class III lanthipeptide synthetase MicKC, and mutation of the conserved catalytic residues abolished the production of dehydrated product . Similar results were reported from in vitro reconstitution of the biosynthesis of another thiamitide termed thioholgamide . In the current study, adopting the nomenclature from the lxm BGC, we first replaced the original numerical order-based designation of genes in the cao BGC to correspond to their lxm homologs (Figure B).…”

Section: Resultssupporting

confidence: 54%

“… 12 Similar results were reported from in vitro reconstitution of the biosynthesis of another thiamitide termed thioholgamide. 14 In the current study, adopting the nomenclature from the lxm BGC, 4 we first replaced the original numerical order-based designation of genes in the cao BGC to correspond to their lxm homologs ( Figure 1 B). To verify the kinase activity of CaoK, we co-expressed CaoK with the N-terminally His 6 -tagged precursor peptide CaoA (His 6 –CaoA) in E. coli .…”

Section: Resultsmentioning

confidence: 99%

“…Similar results were very recently reported for the dehydratase composed of ThoCD involved in thioholgamide biosynthesis. 14 Size exclusion chromatography (SEC) purification indicated the formation of a heterodimer composed of His 6 –CaoK and CaoY. The precursor peptide CaoA was then reacted with the purified CaoK–CaoY complex in the presence of adenosine triphosphate (ATP) and MgCl 2 , resulting in 8-fold dehydrated CaoA as the main product ( Figure 2 A).…”

Section: Resultsmentioning

confidence: 99%

“…This predicted structural similarity was also reported recently for ThoD involved in thioholgamide biosynthesis. 14 Lys136 (Lys219 in CaoY) and Tyr158 (Tyr236 in CaoY) of SpvC that are involved in deprotonation of the α-proton of pSer/pThr residues as revealed by structural studies 20 , 25 are highly conserved in all three protein families, whereas Lys104 and His106 are missing in CaoY and its homologues. His106 acts as a catalytic acid to facilitate C β -OP cleavage in OspF/SpvC and LanKC/L ( Figure 3 B).…”

Section: Resultsmentioning

confidence: 99%

“…Our study on the dehydratase of cacaoidin biosynthesis paves the way for further investigation of class V lanthipeptide biosynthesis as well as the biosynthesis of thioamitides that use a similar complex for dehydration. 12 , 14 …”

Section: Discussionmentioning

confidence: 99%

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Mechanistic Studies on Dehydration in Class V Lanthipeptides

et al. 2022

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Lanthipeptides are ribosomally synthesized and post-translationally modified peptides characterized by lanthionine (Lan) and/or methyllanthionine (MeLan) residues. Four classes of enzymes have been identified to install these structures in a substrate peptide. Recently, a novel class of lanthipeptides was discovered that lack genes for known class I–IV lanthionine synthases in their biosynthetic gene cluster (BGC). In this study, the dehydration of Ser/Thr during the biosynthesis of the class V lanthipeptide cacaoidin was reconstituted in vitro . The aminoglycoside phosphotransferase-like enzyme CaoK iteratively phosphorylates Ser/Thr residues on the precursor peptide CaoA, followed by phosphate elimination catalyzed by the HopA1 effector-like protein CaoY to achieve eight successive dehydrations. CaoY shows sequence similarity to the OspF family proteins and the lyase domains of class III/IV lanthionine synthetases, and mutagenesis studies identified residues that are critical for catalysis. An AlphaFold prediction of the structure of the dehydration enzyme complex engaged with its substrate suggests the importance of hydrophobic interactions between the CaoA leader peptide and CaoK in enzyme–substrate recognition. This model is supported by site-directed mutagenesis studies.

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Section: Resultssupporting

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Mechanistic Studies on Dehydration in Class V Lanthipeptides

et al. 2022

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Novel Biocatalysts from Specialized Metabolism

Kries,

Trottmann,

Hertweck

2023

Angewandte Chemie

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Enzymes are increasingly recognized as valuable (bio)catalysts that complement existing synthetic methods. However, the range of biotransformations used in the laboratory is limited. Here we give an overview on the biosynthesis‐inspired discovery of novel biocatalysts that address various synthetic challenges. Prominent examples from this dynamic field highlight remarkable enzymes for protecting‐group‐free amide formation and modification, control of pericyclic reactions, achieve stereoselective hetero‐ and polycyclizations, enable atroposelective aryl couplings, facilitate site‐selective C‐H activations, introduce ring strain, and promote N‐N bond formations. We also explore unusual functions of cytochrome P450 monooxygenases, radical SAM‐dependent enzymes, flavoproteins, and enzymes recruited from primary metabolism, which offer opportunities for synthetic biology, enzyme engineering, directed evolution, and catalyst design.

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An Enzymatic Prodrug‐like Route to Thio and Selenoamides

Ishida,

Litomska,

Dunbar

et al. 2024

Angewandte Chemie

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6‐Thioguanine (6TG) is a clinically used antitumor agent that was rationally designed as a DNA‐targeting antimetabolite, but it also occurs naturally. 6TG is a critical virulence factor produced by Erwinia amylovorans, a notorious plant pathogen that causes fire blight of pome fruit trees. The biosynthesis of the rare thioamide metabolite involves an adenylating enzyme (YcfA) and a sulfur‐mobilizing enzyme (YcfC), but the mechanism of sulfur transfer and putative intermediates have remained elusive. Through dissection and in vitro reconstitution of the thionation process using diverse substrates, we uncover an intermediate, prodrug‐like thio‐conjugate and elucidate the precise enzyme functions. YcfA not only adenylates GMP but also transfers the mercapto group of l‐cysteine to the activated carbonyl. A designated C‐S lyase (YcfC) then cleaves the resulting S‐adduct to yield the thioamide. This pathway is distinct from canonical tRNA sulfur modifications and known enzymatic peptide thionations. By exploring a wide range of substrate surrogates, we exploited the tolerance of the enzyme pair to produce even a seleno analogue. This study provides valuable insight into a previously unexplored area of bacterial thioamide formation and lays the groundwork for synthetic biology approaches to produce thioamide antimetabolites.

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Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

Cited by 23 publications

References 49 publications

Mechanistic Studies on Dehydration in Class V Lanthipeptides

Mechanistic Studies on Dehydration in Class V Lanthipeptides

Novel Biocatalysts from Specialized Metabolism

An Enzymatic Prodrug‐like Route to Thio and Selenoamides

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