Breaking Cyclic Dipeptide Prenyltransferase Regioselectivity by Unnatural Alkyl Donors

The behavior of five cyclic dipeptide prenyltransferases, responsible for C2-regular, C2-reverse, or C3-reverse prenylation, was investigated in the presence of the unnatural alkyl donors monomethylallyl and 2-pentenyl diphosphate. Both substrates were well accepted by the tested enzymes. Interestingly, C2-reverse and C3-reverse monoalkylated derivatives were identified as enzyme products in all of the enzyme assays. These findings indicate their similar reaction characteristics in the presence of unnatural alkyl donors

K–H₃C and K–Sn Interactions in Potassium Trimethylstannyl Complexes: A Structural, Mechanochemical, and NMR Study

A series of trimethylstannyl potassium complexes [K­(L)­SnMe₃] with different auxiliary ligands L (L = 18-C-6, (TMEDA)₂ (TMEDA = tetramethylethylenediamine), and (12-C-4)₂) were synthesized by alkoxide-induced B–Sn bond cleavage. X-ray structure determinations were performed for all these complexes, and the structural chemistry was studied in detail. For L = 18-C-6 and (TMEDA)₂ the solid state structures comprise polymeric [K­(L)­SnMe₃]_<i>n</i> chains containing bidentate trimethylstannyl anions bridging two [K­(L)]⁺ ions, featuring unsymmetrical coordination of the [K­(L)]⁺ ion by K–Sn and K–H₃C interactions as a central structural motif. In contrast, for L = (12-C-4)₂, separated [K­(12-C-4)₂]⁺ and [SnMe₃]⁻ ions are observed. Unexpectedly, in the presence of tetrahydrofuran (THF), [K­(18-C-6)­SnMe₃]_<i>n</i> forms upon crystallization a new species consisting of separated [K­(18-C-6)­(THF)₂]⁺ and [(Me₂SnCH₃)­K­(18-C-6)­SnMe₃]⁻ ions. In this unsymmetrical anion two trimethylstannyl anions coordinate a single [K­(18-C-6)]⁺ ion; one trimethylstannyl anion coordinates via a K–Sn interaction, and the second coordinates via a K–H₃C interaction. Simulations of the mechanochemical properties (compliance constants) applying approximated density functional theory revealed that both interactions are very soft and are of comparable strength. Moreover, according to our gas phase simulations the unsymmetrically coordinated [(Me₂SnCH₃)­K­(18-C-6)­SnMe₃]⁻ is indeed thermodynamically favored over both possible symmetrical isomers with either K–Sn or K–H₃C coordination. Furthermore, the existence of multiple species due to the two coordination modes and aggregates of [K­(18-C-6)­SnMe₃] in solution is suggested by NMR spectroscopic studies using ¹H, NOESY/ROESY, and ¹H pulsed field gradient diffusion experiments

K–H₃C and K–Sn Interactions in Potassium Trimethylstannyl Complexes: A Structural, Mechanochemical, and NMR Study

A series of trimethylstannyl potassium complexes [K­(L)­SnMe₃] with different auxiliary ligands L (L = 18-C-6, (TMEDA)₂ (TMEDA = tetramethylethylenediamine), and (12-C-4)₂) were synthesized by alkoxide-induced B–Sn bond cleavage. X-ray structure determinations were performed for all these complexes, and the structural chemistry was studied in detail. For L = 18-C-6 and (TMEDA)₂ the solid state structures comprise polymeric [K­(L)­SnMe₃]_<i>n</i> chains containing bidentate trimethylstannyl anions bridging two [K­(L)]⁺ ions, featuring unsymmetrical coordination of the [K­(L)]⁺ ion by K–Sn and K–H₃C interactions as a central structural motif. In contrast, for L = (12-C-4)₂, separated [K­(12-C-4)₂]⁺ and [SnMe₃]⁻ ions are observed. Unexpectedly, in the presence of tetrahydrofuran (THF), [K­(18-C-6)­SnMe₃]_<i>n</i> forms upon crystallization a new species consisting of separated [K­(18-C-6)­(THF)₂]⁺ and [(Me₂SnCH₃)­K­(18-C-6)­SnMe₃]⁻ ions. In this unsymmetrical anion two trimethylstannyl anions coordinate a single [K­(18-C-6)]⁺ ion; one trimethylstannyl anion coordinates via a K–Sn interaction, and the second coordinates via a K–H₃C interaction. Simulations of the mechanochemical properties (compliance constants) applying approximated density functional theory revealed that both interactions are very soft and are of comparable strength. Moreover, according to our gas phase simulations the unsymmetrically coordinated [(Me₂SnCH₃)­K­(18-C-6)­SnMe₃]⁻ is indeed thermodynamically favored over both possible symmetrical isomers with either K–Sn or K–H₃C coordination. Furthermore, the existence of multiple species due to the two coordination modes and aggregates of [K­(18-C-6)­SnMe₃] in solution is suggested by NMR spectroscopic studies using ¹H, NOESY/ROESY, and ¹H pulsed field gradient diffusion experiments

Expansion of Enzymatic Friedel–Crafts Alkylation on Indoles: Acceptance of Unnatural β‑Unsaturated Allyl Diphospates by Dimethylallyl-tryptophan Synthases

Prenyltransferases of the dimethylallyl-tryptophan synthase (DMATS) superfamily catalyze Friedel–Crafts alkylation with high flexibility for aromatic substrates, but the high specificity for dimethylallyl diphosphate (DMAPP) prohibits their application as biocatalysts. We demonstrate here that at least one methyl group in DMAPP can be deleted or shifted to the δ-position. For acceptance by some DMATS enzymes, however, a double bond must be situated at the β-position. Furthermore, the alkylation position of an analogue can differ from that of DMAPP

Coupling of Guanine with <i>cyclo</i>-l‑Trp‑l‑Trp Mediated by a Cytochrome P450 Homologue from <i>Streptomyces purpureus</i>

A cyclo-l-Trp-l-Trp tailoring P450 with novel function from <i>Streptomyces purpureus</i> was identified by heterologous expression in <i>S. coelicolor</i> and in vitro assays the recombinant protein. Structural elucidation revealed that this enzyme catalyzes the transfer of a guanine moiety to the indole ring of the cyclodipeptide via a C–N bond. Adduct products of CDP and guanine are unprecedented in nature, and CDP modification by coupling with guanine has not been reported prior to this study

A <i>Streptomyces</i> Cytochrome P450 Enzyme Catalyzes Regiospecific <i>C</i>2‑Guaninylation for the Synthesis of Diverse Guanitrypmycin Analogs

Heterologous expression of a cdps-p450 locus from Streptomyces sp. NRRL S-1521 led to the identification of guanitrypmycin D1, a new guaninylated diketopiperazine. The cytochrome P450 GutD1521 catalyzed the regiospecific transfer of guanine to C-2 of the indole ring of cyclo-(l-Trp-l-Tyr) via a C–C linkage and represents a new chemical transformation within this enzyme class. Furthermore, GutD1521 efficiently accepts several other tryptophan-containing cyclodipeptides or derivatives for regiospecific coupling with guanine, thus generating different guanitrypmycin analogs

Complementary Flavonoid Prenylations by Fungal Indole Prenyltransferases

Flavonoids are found mainly in plants and exhibit diverse biological and pharmacological activities, which can often be enhanced by prenylations. In plants, such reactions are catalyzed by membrane-bound prenyltransferases. In this study, the prenylation of nine flavonoids from different classes by a soluble fungal prenyltransferase (AnaPT) involved in the biosynthesis of the prenylated indole alkaloid acetylaszonalenin is demonstrated. The behavior of AnaPT toward flavonoids regarding substrate acceptance and prenylation positions clearly differs from that of the indole prenyltransferase 7-DMATS. The two enzymes are therefore complementary in flavonoid prenylations

Caulosegnins I–III: A Highly Diverse Group of Lasso Peptides Derived from a Single Biosynthetic Gene Cluster

Lasso peptides are natural products of ribosomal origin with a unique knotted structural fold. Even though only a few of them are known, recent reports of newly isolated lasso peptides were scarce. In this work, we report the identification of a novel lasso peptide gene cluster from <i>Caulobacter segnis</i>, that produces three new lasso peptides (caulosegnins I, II, and III) using a single biosynthetic machinery. These lasso peptides possess different ring sizes and amino acid sequences. In this study, we have developed a system for enhanced lasso peptide production to allow isolation of these compounds through heterologous expression in <i>Escherichia coli</i>. We were able to elucidate the structure of the most abundant lasso peptide caulosegnin I via NMR spectroscopic analysis and performed a thorough mutational analysis that gave insight into their biosynthesis and revealed important factors affecting the stabilization of the lasso fold in general. The caulosegnins also show a diverse behavior when subjected to thermal denaturation, which is exceptional as all lasso peptides were believed to have an intrinsic high thermal stability

Lasso Peptides: An Intriguing Class of Bacterial Natural Products

ConspectusNatural products of peptidic origin often represent a rich source of medically relevant compounds. The synthesis of such polypeptides in nature is either initiated by deciphering the genetic code on the ribosome during the translation process or driven by ribosome-independent processes. In the latter case, highly modified bioactive peptides are assembled by multimodular enzymes designated as nonribosomal peptide synthetases (NRPS) that act as a protein-template to generate chemically diverse peptides. On the other hand, the ribosome-dependent strategy, although relying strictly on the 20–22 proteinogenic amino acids, generates structural diversity by extensive post-translational-modification. This strategy seems to be highly distributed in all kingdoms of life. One example for this is the lasso peptides, which are an emerging class of ribosomally assembled and post-translationally modified peptides (RiPPs) from bacteria that were first described in 1991.A wide range of interesting biological activities are known for these compounds, including antimicrobial, enzyme inhibitory, and receptor antagonistic activities. Since 2008, genome mining approaches allowed the targeted isolation and characterization of such molecules and helped to better understand this compound class and their biosynthesis. Their defining structural feature is a macrolactam ring that is threaded by the C-terminal tail and held in position by sterically demanding residues above and below the ring, resulting in a unique topology that is reminiscent of a lariat knot. The ring closure is achieved by an isopeptide bond formed between the N-terminal α-amino group of a glycine, alanine, serine, or cysteine and the carboxylic acid side chain of an aspartate or glutamate, which can be located at positions 7, 8, or 9 of the amino acid sequence.In this Account, we discuss the newest findings about these compounds, their biosynthesis, and their physicochemical properties. This includes the suggested mechanism through which the precursor peptide is enzymatically processed into a mature lasso peptide and crucial residues for enzymatic recognition. Furthermore, we highlight new insights considering the protease and thermal stability of lasso peptides and discuss why seven amino acid residue rings are likely to be the lower limit feasible for this compound class. To elucidate their fascinating three-dimensional structures, NMR spectroscopy is commonly employed. Therefore, the general methodology to elucidate these structures by NMR will be discussed and pitfalls for these approaches are highlighted. In addition, new tools provided by recent investigations to assess and prove the lasso topology without a complete structure elucidation will be summarized. These include techniques like ion mobility–mass spectrometry and a combined approach of thermal and carboxypeptidase treatment with subsequent LC-MS analysis. Nevertheless, even though much was learned about these compounds in recent years, their true native function and the exact enzymatic mechanism of their maturation remain elusive