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1.
New Phytol ; 241(1): 394-408, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36866975

RESUMO

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae. Using activity-based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active ß-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.


Assuntos
Quitinases , Hidrolases , Proteômica , Nicotiana , Pseudomonas syringae , Doenças das Plantas/microbiologia
2.
Proc Natl Acad Sci U S A ; 117(29): 17409-17417, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616567

RESUMO

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.


Assuntos
Peptídeo Hidrolases/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteólise , Solanum lycopersicum/metabolismo , Cladosporium , Solanum lycopersicum/genética , Peptídeo Hidrolases/genética , Phytophthora infestans , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Virulência
3.
Plant J ; 106(6): 1523-1540, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33768644

RESUMO

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.


Assuntos
Aminoidrolases/metabolismo , Arabidopsis/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hipocótilo/efeitos dos fármacos , Aldeído Oxidase/genética , Aldeído Oxidase/metabolismo , Aminoidrolases/genética , Apomorfina/análogos & derivados , Apomorfina/farmacologia , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Herbicidas/farmacologia , Hipocótilo/crescimento & desenvolvimento , Ácidos Indolacéticos , Estrutura Molecular , Picloram/farmacologia , Relação Estrutura-Atividade , Transcriptoma/efeitos dos fármacos
4.
New Phytol ; 235(3): 1287-1301, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35510806

RESUMO

Plants encode > 100 metalloproteases representing > 19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant research. We describe the use of hydroxamate-based photoaffinity probes to explore plant proteomes for metalloproteases. We detected labelling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to nine different metalloprotease families and localize to different subcellular compartments. The probes identified several chloroplastic FtsH proteases, vacuolar aspartyl aminopeptidase DAP1, peroxisomal metalloprotease PMX16, extracellular matrix metalloproteases and many cytosolic metalloproteases. We also identified nonproteolytic metallohydrolases involved in the release of auxin and in the urea cycle. Studies on tobacco plants (Nicotiana benthamiana) infected with the bacterial plant pathogen Pseudomonas syringae uncovered the induced labelling of PRp27, a secreted protein with implicated metalloprotease activity. PRp27 overexpression increases resistance, and PRp27 mutants lacking metal binding site are no longer labelled, but still show increased immunity. Collectively, these studies reveal the power of broad-range metalloprotease profiling in plants using hydroxamate-based probes.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Metaloproteínas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metaloproteases/metabolismo , Metaloproteínas/metabolismo , Doenças das Plantas , Pseudomonas syringae/metabolismo , Nicotiana/metabolismo
5.
Plant Physiol ; 180(4): 1848-1859, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31138623

RESUMO

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays), liverwort (Marchantia polymorpha), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in apx1 mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/metabolismo , Ascorbato Peroxidases/metabolismo , Arabidopsis/genética , Ascorbato Peroxidases/genética , Atrazina/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Hepatófitas/genética , Hepatófitas/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Oryza/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Zea mays/genética , Zea mays/metabolismo
6.
Chemistry ; 26(39): 8524-8531, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32250484

RESUMO

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Omura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Produtos Biológicos/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Produtos Biológicos/química , Estrutura Molecular
7.
Nat Chem Biol ; 10(10): 830-6, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25129030

RESUMO

Jasmonates are lipid-derived plant hormones that regulate plant defenses and numerous developmental processes. Although the biosynthesis and molecular function of the most active form of the hormone, (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), have been unraveled, it remains poorly understood how the diversity of bioactive jasmonates regulates such a multitude of plant responses. Bioactive analogs have been used as chemical tools to interrogate the diverse and dynamic processes of jasmonate action. By contrast, small molecules impairing jasmonate functions are currently unknown. Here, we report on jarin-1 as what is to our knowledge the first small-molecule inhibitor of jasmonate responses that was identified in a chemical screen using Arabidopsis thaliana. Jarin-1 impairs the activity of JA-Ile synthetase, thereby preventing the synthesis of the active hormone, JA-Ile, whereas closely related enzymes are not affected. Thus, jarin-1 may serve as a useful chemical tool in search for missing regulatory components and further dissection of the complex jasmonate signaling networks.


Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Arabidopsis/enzimologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas , Nucleotidiltransferases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Inibidores Enzimáticos/síntese química , Estrutura Molecular , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Oxilipinas/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-Atividade
8.
Plant Cell ; 22(12): 4045-66, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21193570

RESUMO

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Biopolímeros/biossíntese , Carotenoides/biossíntese , Pólen , Policetídeo Sintases/genética , Alelos , Genes de Plantas , Hibridização In Situ , Cinética , Microscopia Eletrônica de Transmissão , Mutação , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Curr Opin Chem Biol ; 73: 102253, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36689818

RESUMO

Platelets are small anucleate cell fragments (2-4 µm in diameter) in the blood, which play an essential role in thrombosis and hemostasis. Genetic or acquired platelet dysfunctions are linked to bleeding, increased risk of thromboembolic events and cardiovascular diseases. Advanced proteomic approaches may pave the way to a better understanding of the roles of platelets in hemostasis, and pathophysiological processes such as inflammation, metastatic spread and thrombosis. Further insights into the molecular biology of platelets are crucial to aid drug development and identify diagnostic markers of platelet activation. Platelet activation is known to be an extremely rapid process and involves multiple post-translational mechanisms at sub second time scale, including proteolysis and phosphorylation. Multi-omics technologies and biochemical approaches can be exploited to precisely probe and define these posttranslational pathways. Notably, the absence of a nucleus in platelets significantly reduces the number of present proteins, simplifying mass spectrometry-based proteomics and metabolomics approaches.


Assuntos
Plaquetas , Trombose , Humanos , Plaquetas/metabolismo , Plaquetas/patologia , Proteômica , Multiômica , Ativação Plaquetária , Trombose/metabolismo , Trombose/patologia
10.
Plant Physiol ; 155(1): 477-89, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21045122

RESUMO

Syringolin A (SylA) is a nonribosomal cyclic peptide produced by the bacterial pathogen Pseudomonas syringae pv syringae that can inhibit the eukaryotic proteasome. The proteasome is a multisubunit proteolytic complex that resides in the nucleus and cytoplasm and contains three subunits with different catalytic activities: ß1, ß2, and ß5. Here, we studied how SylA targets the plant proteasome in living cells using activity-based profiling and imaging. We further developed this technology by introducing new, more selective probes and establishing procedures of noninvasive imaging in living Arabidopsis (Arabidopsis thaliana) cells. These studies showed that SylA preferentially targets ß2 and ß5 of the plant proteasome in vitro and in vivo. Structure-activity analysis revealed that the dipeptide tail of SylA contributes to ß2 specificity and identified a nonreactive SylA derivative that proved essential for imaging experiments. Interestingly, subcellular imaging with probes based on epoxomicin and SylA showed that SylA accumulates in the nucleus of the plant cell and suggests that SylA targets the nuclear proteasome. Furthermore, subcellular fractionation studies showed that SylA labels nuclear and cytoplasmic proteasomes. The selectivity of SylA for the catalytic subunits and subcellular compartments is discussed, and the subunit selectivity is explained by crystallographic data.


Assuntos
Imageamento Tridimensional/métodos , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Pseudomonas syringae/química , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cristalografia por Raios X , Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Peptídeos Cíclicos/química , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pseudomonas syringae/efeitos dos fármacos , Reprodutibilidade dos Testes , Coloração e Rotulagem , Relação Estrutura-Atividade
11.
iScience ; 25(11): 105247, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36339259

RESUMO

Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.

12.
Nat Chem ; 14(1): 15-24, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34903857

RESUMO

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.


Assuntos
Inibidores de beta-Lactamases/farmacologia , beta-Lactamas/metabolismo , Animais , Bactérias Gram-Negativas/efeitos dos fármacos , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Ligação Proteica , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/metabolismo
13.
Nat Prod Rep ; 28(11): 1854-67, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21904761

RESUMO

Syrbactin is a subordinate term for the syringolin, glidobactin and cepafungin natural product families. Their grouping is based on their related molecular frameworks, similar biosynthesis pathways and, most importantly, identical modes-of-action, being irreversible proteasome inhibition. With this report, we aim to review their chemical biology, describing their common, but also differential characteristics.


Assuntos
Produtos Biológicos/química , Peptídeos Cíclicos/química , Inibidores de Proteassoma , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Humanos , Estrutura Molecular , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Ureia/análogos & derivados
15.
Chem Asian J ; 11(20): 2859-2862, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27562692

RESUMO

Regioselective hydrolysis, transesterification, and aminolysis of unactivated, highly substituted pyridine esters were realized under mild conditions by employing neighboring group assisted catalysis. Excellent yields were achieved without active removal of the alcohol byproduct. Regioselective aminolysis had a considerable substrate scope ([hetero]aryl, alkyl and amino acid). A mechanism involving assistance by the deprotonated phenolic OH-group is suggested for hydrolysis and transesterification.

16.
Org Lett ; 13(17): 4518-21, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21812451

RESUMO

The enzymatic activation of 3,4-dehydrolysine and subsequent formation of the 12-membered syringolin macrolactam were investigated. The timing of the desaturation was elucidated through the analysis of the initial adenylation domain of SylD. The SylD-TTE didomain was characterized and demonstrated to be the catalyst for formation of 12-membered macrocycles. When the SylD thioesterase domain was reacted with a family of acyclic CoA both natural and unnatural macrocycles were generated.


Assuntos
Peptídeos Cíclicos/biossíntese , Tioléster Hidrolases/metabolismo , Ureia/análogos & derivados , Estrutura Molecular , Peptídeos Cíclicos/química , Estereoisomerismo , Tioléster Hidrolases/química , Ureia/química
17.
Chem Commun (Camb) ; 47(1): 385-7, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-20830349

RESUMO

The preparation of a Syringolin A/Glidobactin A hybrid (SylA-GlbA) consisting of a SylA macrocycle connected to the GlbA side chain and its potent proteasome targeting of all three proteasomal subsites is reported. The influence of the syrbactin macrocycle moiety on subsite selectivity is demonstrated.


Assuntos
Produtos Biológicos/química , Peptídeos Cíclicos/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Sítios de Ligação/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Chem Biol ; 17(10): 1077-83, 2010 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-21035730

RESUMO

Glidobactins are hybrid NRPS-PKS natural products that function as irreversible proteasome inhibitors. A variety of medium chain 2(E),4(E)-diene fatty acids N-acylate the peptidolactam core and contribute significantly to the potency of proteasome inhibition. We have expressed the initiation NRPS module GlbF (C-A-T) in Escherichia coli and observe soluble active protein only on coexpression with the 8 kDa MbtH-like protein, GlbE. Following adenylation and installation of Thr as a T-domain thioester, the starter condensation domain utilizes fatty acyl-CoA donors to acylate the Thr(1) amino group and generate the fatty acyl-Thr(1)-S-pantetheinyl-GlbF intermediate to be used in subsequent chain elongation. Previously proposed to be mediated via acyl carrier protein fatty acid donors, direct utilization of fatty acyl-CoA donors for N-acylation of T-domain tethered amino acids is likely a common strategy for chain initiation in NRPS-mediated lipopeptide biosynthesis.


Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acilação , Proteínas de Bactérias/genética , Burkholderia/enzimologia , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptídeos Cíclicos/biossíntese , Estrutura Terciária de Proteína
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