Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Methods Enzymol ; 699: 447-475, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38942514

RESUMO

Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.


Assuntos
Alquil e Aril Transferases , Terpenos , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/química , Terpenos/metabolismo , Terpenos/química , Ciclização , Vanádio/metabolismo , Vanádio/química , Especificidade por Substrato , Peroxidases/metabolismo , Peroxidases/química , Peroxidases/genética , Ensaios Enzimáticos/métodos
2.
Org Lett ; 26(27): 5725-5730, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38934639

RESUMO

Halogenated phenazine meroterpenoids are a structurally unusual family of marine actinobacterial natural products that exhibit antibiotic, antibiofilm, and cytotoxic bioactivities. Despite a lack of established phenazine halogenation biochemistry, genomic analysis of Streptomyces sp. CNZ-289, a prolific lavanducyanin and C2-halogenated derivative producer, suggested the involvement of vanadium-dependent haloperoxidases. We subsequently discovered lavanducyanin halogenase (LvcH), characterized it in vitro as a regioselective vanadium-dependent chloroperoxidase, and applied it in late-stage chemoenzymatic synthesis.


Assuntos
Cloreto Peroxidase , Halogenação , Vanádio , Cloreto Peroxidase/metabolismo , Cloreto Peroxidase/química , Vanádio/química , Estrutura Molecular , Streptomyces/química , Estereoisomerismo , Fenazinas/química , Fenazinas/farmacologia , Fenazinas/síntese química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química
3.
ACS Chem Biol ; 18(12): 2457-2463, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38047879

RESUMO

FeII/α-ketoglutarate-dependent dioxygenases (Fe/αKG) make up a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/αKG homologues catalyze an array of chemically useful reactions, hydroxylation typically predominates. Microalgal DabC uniquely forms a novel C-C bond to construct the bioactive pyrrolidine ring in domoic acid biosynthesis; however, we have identified that this kainoid synthase exclusively performs a stereospecific hydroxylation reaction on its cis substrate regioisomer. Mechanistic and kinetic analyses with native and alternative substrates identified a 20-fold rate increase in DabC radical cyclization over ß-hydroxylation with no observable 1,5-hydrogen atom transfer. Moreover, this dual activity was conserved among macroalgal RadC1 and KabC homologues and provided insight into substrate recognition and reactivity trends. Investigation of this substrate-dependent chemistry improves our understanding of kainoid synthases and their biocatalytic application.


Assuntos
Oxigenases de Função Mista , Oxigenases de Função Mista/metabolismo , Hidroxilação , Ciclização , Catálise , Biocatálise
4.
ACS Catal ; 13(14): 9817-9828, 2023 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-37497377

RESUMO

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cyanobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA l-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5'-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated l-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate l-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopy, stable isotope labeling techniques, and X-ray crystallography structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and ß-positions of its substrate before catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate-bound GntC structures and activity assays on site-specific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enable an improved understanding of how nature divergently produces cyclic arginine ncAAs and generate additional tools for their biocatalytic production and downstream biological applications.

5.
bioRxiv ; 2023 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-36993528

RESUMO

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5'-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively γ-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the α- and ß-positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate bound GntC structures and activity assays on sitespecific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications.

6.
J Am Chem Soc ; 144(21): 9372-9379, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35583956

RESUMO

Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.


Assuntos
Cianobactérias , Cianobactérias/genética , Cianobactérias/metabolismo , Toxinas de Cianobactérias , Monitoramento Ambiental , Água Doce , Família Multigênica
7.
mSystems ; 6(4): e0078021, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34427499

RESUMO

The chemical diversity of natural products is established by an elegant network of biosynthetic machinery and controlled by a suite of intracellular and environmental cues. Advances in genomics, transcriptomics, and metabolomics have provided useful insight to understand how organisms respond to abiotic and biotic factors to adjust their chemical output; this has permitted researchers to begin asking bigger-picture questions regarding the ecological significance of these molecules to the producing organism and its community. Our lab is motivated by understanding how select microbes construct and manipulate bioactive molecules by utilizing vanadium-dependent haloperoxidase (VHPO) enzymology. This commentary will give perspective into our efforts to understand the unique VHPO-catalyzed conversions which modulate the activities within two ecologically relevant natural product families. Through enhancing our knowledge of microbial natural product biosynthesis, we can understand how and why these bioactive molecules are created.

8.
Clin Immunol ; 226: 108713, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33711450

RESUMO

Current chemical therapies for Chagas Disease (CD) lack ability to clear Trypanosoma cruzi (Tc) parasites and cause severe side effects, making search for new strategies extremely necessary. We evaluated the action of Tityus serrulatus venom (TsV) components during Tc infection. TsV treatment increased nitric oxide and pro-inflammatory cytokine production by Tc-infected macrophages (MØ), decreased intracellular parasite replication and trypomastigotes release, also triggering ERK1/2, JNK1/2 and p38 activation. Ts7 demonstrated the highest anti-Tc activity, inducing high levels of TNF and IL-6 in infected MØ. TsV/Ts7 presented synergistic effect on p38 activation when incubated with Tc antigen. KPP-treatment of MØ also decreased trypomastigotes releasing, partially due to p38 activation. TsV/Ts7-pre-incubation of Tc demonstrated a direct effect on parasite decreasing MØ-trypomastigotes releasing. In vivo KPP-treatment of Tc-infected mice resulted in decreased parasitemia. Summarizing, this study opens perspectives for new bioactive molecules as CD-therapeutic treatment, demonstrating the TsV/Ts7/KPP-trypanocidal and immunomodulatory activity during Tc infection.


Assuntos
Doença de Chagas/tratamento farmacológico , Imunomodulação/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Escorpiões/metabolismo , Animais , Doença de Chagas/metabolismo , Feminino , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Fatores de Necrose Tumoral/metabolismo
9.
Nat Prod Rep ; 37(10): 1334-1366, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32602506

RESUMO

Covering: Up to January 2020Meroterpenoids derived from the polyketide 1,3,6,8-tetrahydroxynaphthalene (THN) are complex natural products produced exclusively by Streptomyces bacteria. These antibacterial compounds include the napyradiomycins, merochlorins, marinones, and furaquinocins and have inspired many attempts at their chemical synthesis. In this review, we highlight the role played by biosynthetic studies in the stimulation of biomimetic and, ultimately, chemoenzymatic total syntheses of these natural products. In particular, the application of genome mining techniques to marine Streptomyces bacteria led to the discovery of unique prenyltransferase and vanadium-dependent haloperoxidase enzymes that can be used as highly selective biocatalysts in fully enzymatic total syntheses, thus overcoming the limitations of purely chemical reagents.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Enzimas/química , Streptomyces/química , Terpenos/química , Antibacterianos/química , Antibacterianos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Estrutura Molecular , Naftoquinonas/síntese química , Streptomyces/genética , Streptomyces/metabolismo , Terpenos/síntese química , Terpenos/metabolismo
10.
J Proteome Res ; 19(8): 3467-3477, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32597192

RESUMO

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.


Assuntos
Venenos de Escorpião , Animais , Proteínas de Ligação ao Cálcio , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Venenos de Escorpião/farmacologia , Transdução de Sinais
11.
mSystems ; 5(3)2020 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-32487740

RESUMO

Using automated genome analysis tools, it is often unclear to what degree genetic variability in homologous biosynthetic pathways relates to structural variation. This hampers strain prioritization and compound identification and can lead to overinterpretation of chemical diversity. Here, we assessed the metabolic potential of Nocardia, an underinvestigated actinobacterial genus that is known to comprise opportunistic human pathogens. Our analysis revealed a plethora of putative biosynthetic gene clusters of various classes, including polyketide, nonribosomal peptide, and terpenoid pathways. Furthermore, we used the highly conserved biosynthetic pathway for nocobactin-like siderophores to investigate how gene cluster differences correlate to structural differences in the produced compounds. Sequence similarity networks generated by BiG-SCAPE (Biosynthetic Gene Similarity Clustering and Prospecting Engine) showed the presence of several distinct gene cluster families. Metabolic profiling of selected Nocardia strains using liquid chromatography-mass spectrometry (LC-MS) metabolomics data, nuclear magnetic resonance (NMR) spectroscopy, and GNPS (Global Natural Product Social molecular networking) revealed that nocobactin-like biosynthetic gene cluster (BGC) families above a BiG-SCAPE threshold of 70% can be assigned to distinct structural types of nocobactin-like siderophores.IMPORTANCE Our work emphasizes that Nocardia represent a prolific source for natural products rivaling better-characterized genera such as Streptomyces or Amycolatopsis Furthermore, we showed that large-scale analysis of biosynthetic gene clusters using similarity networks with high stringency allows the distinction and prediction of natural product structural variations. This will facilitate future genomics-driven drug discovery campaigns.

12.
Proc Natl Acad Sci U S A ; 117(23): 12799-12805, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457155

RESUMO

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.


Assuntos
Alquil e Aril Transferases/química , Diatomáceas/enzimologia , Dimetilaliltranstransferase/química , Ácido Caínico/análogos & derivados , Neurotoxinas/biossíntese , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Diatomáceas/metabolismo , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Ácido Glutâmico/metabolismo , Ácido Caínico/metabolismo , Magnésio/metabolismo , Prenilação , Ligação Proteica
13.
ACS Chem Biol ; 15(4): 1067-1077, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32195572

RESUMO

Alpiniamide A is a linear polyketide produced by Streptomyces endophytic bacteria. Despite its relatively simple chemical structure suggestive of a linear assembly line biosynthetic construction involving a hybrid polyketide synthase-nonribosomal peptide synthetase enzymatic protein machine, we report an unexpected nonlinear synthesis of this bacterial natural product. Using a combination of genomics, heterologous expression, mutagenesis, isotope-labeling, and chain terminator experiments, we propose that alpiniamide A is assembled in two halves and then ligated into the mature molecule. We show that each polyketide half is constructed using orthogonal biosynthetic strategies, employing either cis- or trans-acyl transferase mechanisms, thus prompting an alternative proposal for the operation of this PKS-NRPS.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Policetídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Genômica , Família Multigênica , Peptídeo Sintases/química , Peptídeo Sintases/genética , Domínios Proteicos , Streptomyces/genética , Streptomyces/metabolismo
14.
Harmful Algae ; 92: 101737, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32113603

RESUMO

Anatoxin-a(S) is the most potent natural neurotoxin produced by fresh water cyanobacteria. It is also the least understood and monitored. Although this potent cholinesterase inhibitor was first reported in the 1970s and connected with animal poisonings, the lack of chemical standards and identified biosynthetic genes together with limited diagnostics and acute reactivity of this naturally-occurring organophosphate have limited our understanding of its environmental breadth and human health implications. Anatoxin-a(S) irreversibly inhibits acetylcholinesterase much like other organophosphate agents like paraoxon. It is however often confused with the similarly named anatoxin-a that has a completely different chemical structure, mechanism of action, and biosynthesis. Herein we propose renaming of anatoxin-a(S) to clarify its distinct structure and mechanism and to draw renewed attention to this potent natural poison. We propose the new name guanitoxin (GNT) to emphasize its distinctive guanidino organophosphate chemical structure.


Assuntos
Cianobactérias , Animais , Inibidores da Colinesterase , Água Doce , Humanos , Neurotoxinas , Organofosfatos/toxicidade
15.
ACS Infect Dis ; 6(4): 563-571, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-31906623

RESUMO

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.


Assuntos
Antibacterianos/metabolismo , Vias Biossintéticas/genética , Microbiota/efeitos dos fármacos , Pirrolidinonas/metabolismo , Streptococcus mutans/metabolismo , Simbiose/efeitos dos fármacos , Antibacterianos/biossíntese , Cárie Dentária/microbiologia , Humanos , Boca/microbiologia , Família Multigênica , Policetídeo Sintases/genética , Streptococcus mutans/genética , Streptococcus mutans/patogenicidade , Ácido Tenuazônico/análogos & derivados , Ácido Tenuazônico/metabolismo
16.
Nat Chem ; 11(10): 880-889, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31527851

RESUMO

Colibactin is an assumed human gut bacterial genotoxin, whose biosynthesis is linked to the clb genomic island that has a widespread distribution in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumour formation and enhance the progression of colorectal cancer via cellular senescence and death induced by DNA double-strand breaks (DSBs); however, the chemical basis that contributes to the pathogenesis at the molecular level has not been fully characterized. Here, we report the discovery of colibactin-645, a macrocyclic colibactin metabolite that recapitulates the previously assumed genotoxicity and cytotoxicity. Colibactin-645 shows strong DNA DSB activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also delineate a complete biosynthetic model for colibactin-645, which highlights a unique fate of the aminomalonate-building monomer in forming the C-terminal 5-hydroxy-4-oxazolecarboxylic acid moiety through the activities of both the polyketide synthase ClbO and the amidase ClbL. This work thus provides a molecular basis for colibactin's DNA DSB activity and facilitates further mechanistic study of colibactin-related colorectal cancer incidence and prevention.


Assuntos
Cobre/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Peptídeos/farmacologia , Policetídeos/farmacologia , Cobre/química , Compostos Macrocíclicos/química , Conformação Molecular , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/química , Policetídeos/química
17.
Angew Chem Int Ed Engl ; 58(25): 8394-8399, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-30963655

RESUMO

l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.


Assuntos
Aminoácidos/metabolismo , Antibacterianos/metabolismo , Antidepressivos/metabolismo , Cinurenina/análogos & derivados , Lipopeptídeos/metabolismo , Pró-Fármacos/metabolismo , Aminoácidos/química , Antibacterianos/química , Antidepressivos/química , Arilformamidase/metabolismo , Cristalografia por Raios X , Cinurenina/biossíntese , Cinurenina/química , Lipopeptídeos/química , Modelos Moleculares , Estrutura Molecular , Pró-Fármacos/química , Triptofano Oxigenase/metabolismo
18.
Angew Chem Int Ed Engl ; 58(25): 8454-8457, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995339

RESUMO

Kainic acid, the flagship member of the kainoid family of natural neurochemicals, is a widely used neuropharmacological agent that helped unravel the key role of ionotropic glutamate receptors, including the kainate receptor, in the central nervous system. Worldwide shortages of this seaweed natural product in the year 2000 prompted numerous chemical syntheses, including scalable preparations with as few as six-steps. Herein we report the discovery and characterization of the concise two-enzyme biosynthetic pathway to kainic acid from l-glutamic acid and dimethylallyl pyrophosphate in red macroalgae and show that the biosynthetic genes are co-clustered in genomes of Digenea simplex and Palmaria palmata. Moreover, we applied a key biosynthetic α-ketoglutarate-dependent dioxygenase enzyme in a biotransformation methodology to efficiently construct kainic acid on the gram scale. This study establishes both the feasibility of mining seaweed genomes for their biotechnological prowess.


Assuntos
Ácido Caínico/metabolismo , Rodófitas/química , Ácido Caínico/química , Estrutura Molecular , Rodófitas/metabolismo
19.
Org Biomol Chem ; 17(13): 3416-3423, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30869693

RESUMO

Thiotetronate-containing natural products, including thiolactomycin, thiotetromycin, and thiotetroamide, are potent, broad-spectrum antibacterial compounds that target fatty acid synthesis in bacteria. Natural modifications at the C-5 dialkyl position in this molecular series result in pronounced bioactivity differences. The C-5 acetamide-containing thiotetroamide, which is the more potent antibacterial agent in this family, is biosynthesized from the C-5 ethyl analogue thiotetromycin via a unique two-enzyme process involving the cytochrome P450-amidotransferase enzyme pair TtmP-TtmN. Herein we synthesized a focused library of 17 novel thiotetromycin derivatives differing at the 5-position alkyl substituent to investigate their biological activities and their reactivity towards the hydroxylase TtmP. Although we observed marginal anti-tuberculosis activity, select thiotetromycin analogues showed antibacterial activity against an Escherichia coli ΔtolC strain with IC50 values in a range of 1.9-36 µg mL-1. Additional screening efforts highlighted select thiotetronate analogues as inhibitors of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT), with a unique scaffold compared to previously identified NNMT inhibitors. In vitro assays further showed that the TtmP P450 was capable of resolving racemic substrate mixtures and had modest promiscuity to hydroxylate derivatives with variable alkyl chains; however triple oxidation to a carboxylic acid remained specific for the natural thiotetromycin substrate. The tendency of TtmP to accept a range of unnatural substrates for hydroxylation makes it an interesting target for P450 engineering towards broader applications.


Assuntos
Antranilato Sintase/metabolismo , Antibacterianos/farmacologia , Produtos Biológicos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/efeitos dos fármacos , Transferases de Grupos Nitrogenados/metabolismo , Antibacterianos/biossíntese , Antibacterianos/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/metabolismo , Tiofenos/farmacologia
20.
Eur J Med Chem ; 166: 119-124, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30690406

RESUMO

Apelins are human peptide hormones with various physiological activities, including the moderation of cardiovascular, renal, metabolic and neurological function. Their potency is dependent on and limited by proteolytic degradation in the circulatory system. Here we identify human plasma kallikrein (KLKB1) as a protease that cleaves the first three N-terminal amino acids (KFR) of apelin-17. The cleavage kinetics are similar to neprilysin (NEP), which cleaves within the critical 'RPRL'-motif thereby inactivating apelin. The resulting C-terminal 14-mer after KLKB1 cleavage has much lower biological activity, and the presence of its N-terminal basic arginine seems to negate the blood pressure lowering effect. Based on C-terminally engineered apelin analogs (A2), resistant to angiotensin converting enzyme 2 (ACE2), attachment of an N-terminal C16 fatty acid chain (PALMitoylation) or polyethylene glycol chain (PEGylation) minimizes KLKB1 cleavage of the 17-mers, thereby extending plasma half-life while fully retaining biological activity. The N-terminally PEGylated apelin-17(A2) is a highly protease resistant analog, with excellent apelin receptor activation and pronounced blood pressure lowering effect.


Assuntos
Apelina/química , Apelina/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Ácidos Graxos/química , Calicreína Plasmática/metabolismo , Polietilenoglicóis/química , Proteólise , Apelina/farmacologia , Humanos , Estabilidade Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...