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1.
Nat Commun ; 12(1): 5469, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552091

RESUMO

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.


Assuntos
Anticorpos Neutralizantes/farmacologia , COVID-19/tratamento farmacológico , Anticorpos de Domínio Único/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Administração Intranasal , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Epitopos/química , Epitopos/metabolismo , Feminino , Masculino , Mesocricetus , Testes de Neutralização , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Domínio Único/administração & dosagem , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química
2.
Methods Enzymol ; 656: 429-458, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34325794

RESUMO

Backbone N-methylation as a posttranslational modification was recently discovered in a class of ribosomally encoded peptides referred to as borosins. The founding members of the borosins are the omphalotins (A-I), backbone N-methylated, macrocyclic dodecapeptides produced by the mushroom Omphalotus olearius. Omphalotins display a strong and selective toxicity toward the plant parasitic nematode Meloidogyne incognita. The primary product omphalotin A is synthesized via a concerted action of the omphalotin precursor protein (OphMA) and the dual function prolyloligopeptidase/macrocyclase (OphP). OphMA consists of α-N-methyltransferase domain that autocatalytically methylates the core peptide fused to its C-terminus via a clasp domain. Genome mining uncovered over 50 OphMA homologs from the fungal phyla Ascomycota and Basidiomycota. However, the derived peptide natural products have not been described yet, except for lentinulins, dendrothelins and gymnopeptides produced by the basidiomycetes Lentinula edodes, Dendrothele bispora and Gymnopus fusipes, respectively. In this chapter, we describe methods used to isolate and characterize these backbone N-methylated peptides and their precursor proteins both in their original hosts and in the heterologous hosts Escherichia coli and Pichia pastoris. These methods may pave the path for both the discovery of novel borosins with interesting bioactivities. In addition, understanding of borosin biosynthetic pathways may allow setting up a biotechnological platform for the production of pharmaceutical leads for orally available peptide drugs.


Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional , Agaricales , Metilação , Peptídeos/genética , Peptídeos/metabolismo , Saccharomycetales
3.
Nat Commun ; 12(1): 4349, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272394

RESUMO

Bacterial extracellular polysaccharides (EPSs) play critical roles in virulence. Many bacteria assemble EPSs via a multi-protein "Wzx-Wzy" system, involving glycan polymerization at the outer face of the cytoplasmic/inner membrane. Gram-negative species couple polymerization with translocation across the periplasm and outer membrane and the master regulator of the system is the tyrosine autokinase, Wzc. This near atomic cryo-EM structure of dephosphorylated Wzc from E. coli shows an octameric assembly with a large central cavity formed by transmembrane helices. The tyrosine autokinase domain forms the cytoplasm region, while the periplasmic region contains small folded motifs and helical bundles. The helical bundles are essential for function, most likely through interaction with the outer membrane translocon, Wza. Autophosphorylation of the tyrosine-rich C-terminus of Wzc results in disassembly of the octamer into multiply phosphorylated monomers. We propose that the cycling between phosphorylated monomer and dephosphorylated octamer regulates glycan polymerization and translocation.


Assuntos
Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/química , Periplasma/metabolismo , Polissacarídeos Bacterianos/metabolismo , Proteínas Tirosina Quinases/química , Motivos de Aminoácidos , Domínio Catalítico , Microscopia Crioeletrônica , Citoplasma/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Periplasma/química , Fosforilação , Conformação Proteica em alfa-Hélice , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/química , Tirosina/metabolismo
4.
Angew Chem Int Ed Engl ; 60(26): 14319-14323, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33856715

RESUMO

Introduction of α-N-methylated non-proteinogenic amino acids into peptides can improve their biological activities, membrane permeability and proteolytic stability. This is commonly achieved, in nature and in the lab, by assembling pre-methylated amino acids. The more appealing route of methylating amide bonds is challenging. Biology has evolved an α-N-automethylating enzyme, OphMA, which acts on the amide bonds of peptides fused to its C-terminus. Due to the ribosomal biosynthesis of its substrate, the activity of this enzyme towards peptides with non-proteinogenic amino acids has not been addressed. An engineered OphMA, intein-mediated protein ligation and solid-phase peptide synthesis have allowed us to demonstrate the methylation of amide bonds in the context of non-natural amides. This approach may have application in the biotechnological production of therapeutic peptides.


Assuntos
Aminoácidos/metabolismo , Metiltransferases/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas , Amidas/química , Amidas/metabolismo , Aminoácidos/química , Metilação , Metiltransferases/química , Peptídeos/química , Conformação Proteica
6.
J Biol Chem ; 296: 100352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33524389

RESUMO

Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes-C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC-are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Heptoses/metabolismo , Oxirredutases/metabolismo , Racemases e Epimerases/metabolismo , Proteínas de Bactérias/química , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/química , Heptoses/química , Humanos , Oxirredutases/química , Conformação Proteica , Racemases e Epimerases/química , Especificidade por Substrato
7.
Nat Methods ; 18(1): 60-68, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33408403

RESUMO

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.


Assuntos
Microscopia Crioeletrônica/métodos , Lipídeos/química , Complexos Multiproteicos/química , Receptores de GABA-A/química , Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Anticorpos de Domínio Único/química , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica
8.
Curr Opin Struct Biol ; 65: iii-iv, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33176964
10.
Nat Struct Mol Biol ; 27(10): 950-958, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32737466

RESUMO

The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.


Assuntos
Anticorpos Antivirais/química , Betacoronavirus/química , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Adulto , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Chlorocebus aethiops , Reações Cruzadas , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Masculino , Pandemias , Peptidil Dipeptidase A/metabolismo , Conformação Proteica , Domínios Proteicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
11.
Curr Opin Struct Biol ; 65: 79-88, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32653730

RESUMO

The amide bond with its planarity and lack of chemical reactivity is at the heart of protein structure. Chemical methylation of amides is known but was considered too harsh to be accessible to biology. Until last year there was no protein structure in the data bank with an enzymatically methylated amide. The discovery that the natural macrocyclic product, omphalotin is ribosomally synthesized, was not as had been assumed by non-ribosomal peptide synthesis. This was the first definitive evidence that an enzyme could methylate the amide bond. The enzyme, OphMA, iteratively self-hypermethylates its own C-terminus using SAM as cofactor. A second enzyme OphP, a prolyl oligopeptidase cleaves the core peptide from OphMA and cyclizes it into omphalotin. The molecular mechanism for OphMA was elucidated by mutagenesis, structural, biochemical and theoretical studies. This review highlights current progress in peptide N-methylating enzymes.

12.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , COVID-19 , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2 , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
14.
J Biol Chem ; 295(40): 13724-13736, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32669363

RESUMO

The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.


Assuntos
Proteínas de Bactérias/química , Clostridiales/enzimologia , Ácido N-Acetilneuramínico/química , Oxirredutases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridiales/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Teste de Complementação Genética , Humanos , Mucinas/química , Mucinas/metabolismo , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo
15.
ACS Chem Biol ; 15(7): 1901-1912, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32491837

RESUMO

The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.


Assuntos
Proteínas Fúngicas/metabolismo , Metiltransferases/metabolismo , Peptídeos Cíclicos/metabolismo , Precursores de Proteínas/metabolismo , Agaricales/enzimologia , Sequência de Aminoácidos , Metilação , Mutação , Peptídeos Cíclicos/genética , Domínios Proteicos , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional , Especificidade por Substrato
16.
Chem Commun (Camb) ; 56(55): 7617-7620, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32515440

RESUMO

The enzyme ForT catalyzes C-C bond formation between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5'-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis.


Assuntos
Carbono-Carbono Ligases/metabolismo , Fosforribosil Pirofosfato/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Carbono-Carbono Ligases/química , Domínio Catalítico , Cristalografia por Raios X , Modelos Químicos , Fosforribosil Pirofosfato/química , Ligação Proteica , Streptomyces/enzimologia
17.
Angew Chem Int Ed Engl ; 59(15): 6054-6061, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-31903677

RESUMO

Heterocycles, a class of molecules that includes oxazoles, constitute one of the most common building blocks in current pharmaceuticals and are common in medicinally important natural products. The antitumor natural product nataxazole is a model for a large class of benzoxazole-containing molecules that are made by a pathway that is not characterized. We report structural, biochemical, and chemical evidence that benzoxazole biosynthesis proceeds through an ester generated by an ATP-dependent adenylating enzyme. The ester rearranges via a tetrahedral hemiorthoamide to yield an amide, which is a shunt product and not, as previously thought, an intermediate in the pathway. A second zinc-dependent enzyme catalyzes the formation of hemiorthoamide from the ester but, by shuttling protons, the enzyme eliminates water, a reverse hydrolysis reaction, to yield the benzoxazole and avoids the amide. These insights have allowed us to harness the pathway to synthesize a series of novel halogenated benzoxazoles.


Assuntos
Benzoxazóis/química , Benzoxazóis/metabolismo , Ésteres/química , Trifosfato de Adenosina/metabolismo , Enzimas/química , Enzimas/metabolismo , Halogenação , Modelos Moleculares , Conformação Proteica
18.
Nat Rev Microbiol ; 18(3): 164-176, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31792365

RESUMO

Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Enterobacteriaceae/enzimologia , Enterobacteriaceae/metabolismo , Porinas/metabolismo , Antibacterianos/metabolismo , Transporte Biológico , Farmacorresistência Bacteriana , Enterobacteriaceae/efeitos dos fármacos
19.
Chem Commun (Camb) ; 55(96): 14502-14505, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31730149

RESUMO

ForI is a PLP-dependent enzyme from the biosynthetic pathway of the C-nucleoside antibiotic formycin. Cycloserine is thought to inhibit PLP-dependent enzymes by irreversibly forming a PMP-isoxazole. We now report that ForI forms novel PMP-diketopiperazine derivatives following incubation with both d and l cycloserine. This unexpected result suggests chemical diversity in the chemistry of cycloserine inhibition.


Assuntos
Proteínas de Bactérias/metabolismo , Dicetopiperazinas/química , Formicinas/biossíntese , Fosfato de Piridoxal/química , Piridoxamina/análogos & derivados , Transaminases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Ciclosserina/química , Dicetopiperazinas/metabolismo , Formicinas/química , Concentração de Íons de Hidrogênio , Piridoxamina/química , Piridoxamina/metabolismo , Streptomyces/química , Streptomyces/metabolismo , Transaminases/antagonistas & inibidores , Transaminases/genética
20.
Nat Chem ; 11(12): 1091-1097, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31611633

RESUMO

Oceanic cyanobacteria are the most abundant oxygen-generating phototrophs on our planet and are therefore important to life. These organisms are infected by viruses called cyanophages, which have recently shown to encode metabolic genes that modulate host photosynthesis, phosphorus cycling and nucleotide metabolism. Herein we report the characterization of a wild-type flavin-dependent viral halogenase (VirX1) from a cyanophage. Notably, halogenases have been previously associated with secondary metabolism, tailoring natural products. Exploration of this viral halogenase reveals it capable of regioselective halogenation of a diverse range of substrates with a preference for forming aryl iodide species; this has potential implications for the metabolism of the infected host. Until recently, a flavin-dependent halogenase that is capable of iodination in vitro had not been reported. VirX1 is interesting from a biocatalytic perspective as it shows strikingly broad substrate flexibility and a clear preference for iodination, as illustrated by kinetic analysis. These factors together render it an attractive tool for synthesis.


Assuntos
Bacteriófagos/enzimologia , Cianobactérias/virologia , Oxirredutases/metabolismo , Bacteriófagos/genética , Técnicas de Química Sintética , Halogenação , Cinética , Estrutura Molecular , Especificidade por Substrato
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