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1.
Biosci Biotechnol Biochem ; 88(3): 270-275, 2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38169014

RESUMO

Secondary metabolites are specialized metabolic products synthesized by plants, insects, and bacteria, some of which exhibit significant physiological activities against other organisms. Plants containing bioactive secondary metabolites have been used in traditional medicine for centuries. In developed countries, one-fourth of medicines directly contain plant-derived compounds or indirectly contain them via semi-synthesis. These compounds have contributed considerably to the development of not only medicine but also molecular biology. Moreover, the biosynthesis of these physiologically active secondary metabolites has attracted substantial interest and has been extensively studied. However, in many cases, the degradation mechanisms of these secondary metabolites remain unclear. In this review, some unique microbial degradation pathways for lignans and C-glycosides are explored.


Assuntos
Bactérias , Fungos , Glicosídeos , Lignanas , Lignanas/metabolismo , Glicosídeos/metabolismo , Bactérias/metabolismo , Redes e Vias Metabólicas , Fungos/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34583991

RESUMO

C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme "C-glycoside 3-oxidase" named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.


Assuntos
Flavina-Adenina Dinucleotídeo/metabolismo , Glicosídeos/metabolismo , Microbacterium/metabolismo , Glicosídeos Cardíacos/metabolismo , Carmim/metabolismo , Catálise , Redes e Vias Metabólicas/fisiologia , Mutagênese/fisiologia , Oxirredutases/metabolismo , Especificidade por Substrato
3.
Appl Environ Microbiol ; 89(11): e0114523, 2023 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-37874289

RESUMO

IMPORTANCE: Pepper is a spice that has been used worldwide since the Age of Discovery. The substance that is responsible for the spiciness in pepper is piperine, a type of alkaloid. It has never been reported how piperine is degraded by microorganisms. In this study, we discovered a bacterium in the soil that is capable of catabolizing piperine as its sole nitrogen source. Furthermore, we discovered the enzyme involved in piperine metabolism. This enzyme decomposed the methylenedioxyphenyl group, which is the common structure in various plant-derived bioactive compounds such as sesamin, piperonal, safrole, and berberin. By utilizing this enzyme, piperine can be converted into a useful antioxidant compound. The findings about previously unknown metabolic pathways in nature can lead to the discovery of new enzymes and provide methods for the enzymatic synthesis of useful compounds.


Assuntos
Actinobacteria , Alcaloides , Alcamidas Poli-Insaturadas/química , Piperidinas/química
4.
Proc Natl Acad Sci U S A ; 113(32): 9087-92, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27444012

RESUMO

Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, Vmax = 9.3 ± 0.4 (µmol⋅min(-1)⋅mg(-1)), and kcat = 7.9 ± 0.3 s(-1) Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (-)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins.


Assuntos
Dioxóis/metabolismo , Lignanas/metabolismo , Micrococcaceae/metabolismo , Cinética , Micrococcaceae/isolamento & purificação , Mutação , Microbiologia do Solo , Especificidade por Substrato
5.
J Biol Chem ; 291(4): 1735-1750, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26586916

RESUMO

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its "enzymatic activity" and (ii) subsequent "chemical" S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Ligases/metabolismo , Peptídeos/metabolismo , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Carbono-Oxigênio Ligases/química , Carbono-Oxigênio Ligases/genética , Especificidade por Substrato
6.
Proc Natl Acad Sci U S A ; 111(48): 17152-7, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25411318

RESUMO

Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of L-ascorbic acid and L-cysteine as substrates by analytical methods for enzymes. Our findings were as follows: (i) oxidation reactions producing H2O2 proceeded upon addition of ACT to the reaction mixture; (ii) ACT was not consumed during the reactions; and (iii) a small amount (catalytic amount) of ACT consumed an excess amount of the substrates. Even at room temperature, atmospheric pressure, and neutral pH, ACT showed catalytic activity in aqueous solution, and ACT exhibited substrate specificity in the oxidation reactions. These findings reveal ACT to be an organocatalyst. ACT is known to show antibiotic activity, but its mechanism of action remains unknown. On the basis of our results, we propose that ACT kills bacteria by catalyzing the production of toxic levels of H2O2. We also screened various other natural products of bacterial, plant, and animal origins and found that several of the compounds exhibited catalytic activity, suggesting that living organisms produce and use these compounds as biocatalysts in nature.


Assuntos
Produtos Biológicos/metabolismo , Oxirredutases/metabolismo , Streptomyces coelicolor/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Ácido Ascórbico/metabolismo , Produtos Biológicos/química , Catálise , Cromatografia Líquida de Alta Pressão , Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Peso Molecular , Oxirredução , Oxirredutases/química , Especificidade por Substrato , Temperatura
7.
Biosci Biotechnol Biochem ; 80(6): 1230-7, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26923287

RESUMO

An inducible expression vector, pSH19, which harbors regulatory expression system PnitA-NitR, for streptomycetes was constructed previously. Here, we have modified pSH19 to obtain shuttle vectors for Streptomyces-E. coli by introducing the replication origin of a plasmid for E. coli (ColE1) and an antibiotic-resistant gene. Six inducible shuttle vectors, pESH19cF, pESH19cR, pESH19kF, pESH19kR, pESH19aF, and pESH19aR, for Streptomyces-E. coli, were successfully developed. The stability of these vectors was examined in five different E. coli strains and Streptomyces lividans TK24. The stability test showed that the pSH19-derived shuttle vectors were stable in E. coli Stbl2 and S. lividans TK24. Heterologous expression experiments involving each of the catechol 2,3-dioxygenase, nitrilase, and N-substituted formamide deformylase genes as a reporter gene showed that pESH19cF, pESH19kF, and pESH19aF possess inducible expression ability in S. lividans TK24. Thus, these vectors were found to be useful expression tools for experiments on both Gram-negative and Gram-positive bacterial genes.


Assuntos
Aminoidrolases/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Vetores Genéticos/metabolismo , Plasmídeos/metabolismo , Streptomyces lividans/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Aminoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Catecol 2,3-Dioxigenase/genética , Catecol 2,3-Dioxigenase/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Engenharia Genética , Vetores Genéticos/química , Plasmídeos/química , Regiões Promotoras Genéticas , Streptomyces lividans/metabolismo
8.
Nat Chem Biol ; 7(7): 461-8, 2011 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-21642985

RESUMO

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.


Assuntos
Policetídeo Sintases/metabolismo , Piranos/química , Compostos de Espiro/química , Vias Biossintéticas , Cromatografia Líquida de Alta Pressão , Ciclização , Espectrometria de Massas , Estrutura Molecular , Policetídeo Sintases/genética , Estereoisomerismo , Streptomyces/enzimologia , Streptomyces/genética
9.
J Biol Chem ; 285(51): 39663-71, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20937800

RESUMO

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) that exhibits antitumor activity and is produced by the Streptomyces sp. strain KO-3988. Bioinformatic analysis of furaquinocin biosynthesis has identified Fur7 as a possible prenyltransferase that attaches a geranyl group to an unidentified polyketide scaffold. Here, we report the identification of a physiological polyketide substrate for Fur7, as well as its reaction product and the biochemical characterization of Fur7. A Streptomyces albus transformant (S. albus/pWHM-Fur2_del7) harboring the furaquinocin biosynthetic gene cluster lacking the fur7 gene did not produce furaquinocin but synthesized the novel intermediate 2-methoxy-3-methyl-flaviolin. After expression and purification from Escherichia coli, the recombinant Fur7 enzyme catalyzed the transfer of a geranyl group to 2-methoxy-3-methyl-flaviolin to yield 6-prenyl-2-methoxy-3-methyl-flaviolin and 7-O-geranyl-2-methoxy-3-methyl-flaviolin in a 10:1 ratio. The reaction proceeded independently of divalent cations. When 6-prenyl-2-methoxy-3-methyl-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del7, furaquinocin production was restored. The promiscuous substrate specificity of Fur7 was demonstrated with respect to prenyl acceptor substrates and prenyl donor substrates. The steady-state kinetic constants of Fur7 with each prenyl acceptor substrate were also calculated.


Assuntos
Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Dimetilaliltranstransferase/metabolismo , Macrolídeos/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biologia Computacional/métodos , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Escherichia coli/genética , Cinética , Família Multigênica/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/genética
10.
Metab Eng ; 13(6): 629-37, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21835257

RESUMO

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.


Assuntos
Dimetilaliltranstransferase/metabolismo , Glycine max/enzimologia , Lotus/enzimologia , Engenharia Metabólica/métodos , Plantas Geneticamente Modificadas/enzimologia , Polifenóis/biossíntese , Dimetilaliltranstransferase/genética , Flavanonas/administração & dosagem , Lotus/genética , Plantas Geneticamente Modificadas/genética , Prenilação/genética , Sophora/enzimologia , Sophora/genética , Glycine max/genética , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
11.
Biosci Biotechnol Biochem ; 75(3): 505-10, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21389612

RESUMO

We performed combinational bioconversion of substituted naphthalenes with PhnA1A2A3A4 (an aromatic dihydroxylating dioxygenase from marine bacterium Cycloclasticus sp. strain A5) and prenyltransferase NphB (geranyltransferase from Streptomyces sp. strain CL190) or SCO7190 (dimethylallyltransferase from Streptomyces coelicolor A3(2)) to produce prenyl naphthalen-ols. Using 2-methylnaphthalene, 1-methoxynaphthalene, and 1-ethoxynaphthalene as the starting substrates, 10 novel prenyl naphthalen-ols were produced by combinational bioconversion. These novel prenyl naphthalen-ols each showed potent antioxidative activity against a rat brain homogenate model. 2-(2,3-Dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one (2',3'-dihydroxychrysin) generated with another aromatic dihydroxylating dioxygenase and subsequent dehydrogenase was also geranylated at the C-5'-carbon by the action of NphB.


Assuntos
Sistema Livre de Células/metabolismo , Dimetilaliltranstransferase/metabolismo , Dioxigenases/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Animais , Antioxidantes/farmacologia , Biotransformação , Encéfalo/metabolismo , Clonagem Molecular , Dimetilaliltranstransferase/genética , Dioxigenases/genética , Escherichia coli , Expressão Gênica , Naftalenos/química , Piscirickettsiaceae/química , Piscirickettsiaceae/enzimologia , Prenilação , Ratos , Proteínas Recombinantes/genética , Streptomyces/química , Streptomyces/enzimologia
12.
J Gen Appl Microbiol ; 67(1): 24-32, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33162426

RESUMO

Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidroliases/biossíntese , Hidroliases/genética , Pseudomonas chlororaphis/enzimologia , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Ferro , Mutagênese Sítio-Dirigida , Proteínas Recombinantes , Urease/metabolismo
13.
Nat Commun ; 12(1): 6294, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728636

RESUMO

C-Glycosides, in which a sugar moiety is linked via a carbon-carbon (C-C) bond to a non-sugar moiety (aglycone), are found in our food and medicine. The C-C bond is cleaved by intestinal microbes and the resulting aglycones exert various bioactivities. Although the enzymes responsible for the reactions have been identified, their catalytic mechanisms and the generality of the reactions in nature remain to be explored. Here, we present the identification and structural basis for the activation of xenobiotic C-glycosides by heterocomplex C-deglycosylation enzymes from intestinal and soil bacteria. They are found to be metal-dependent enzymes exhibiting broad substrate specificity toward C-glycosides. X-ray crystallographic and cryo-electron microscopic analyses, as well as structure-based mutagenesis, reveal the structural details of these enzymes and the detailed catalytic mechanisms of their remarkable C-C bond cleavage reactions. Furthermore, bioinformatic and biochemical analyses suggest that the C-deglycosylation enzymes are widely distributed in the gut, soil, and marine bacteria.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Trato Gastrointestinal/metabolismo , Glicosídeos/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/química , Cristalografia por Raios X , Trato Gastrointestinal/microbiologia , Glicosídeos/química , Glicosilação , Filogenia , Elementos Estruturais de Proteínas , Homologia de Sequência , Especificidade por Substrato
14.
Nat Commun ; 10(1): 413, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679427

RESUMO

Although cyclic imines are present in various bioactive secondary metabolites, their degradative metabolism remains unknown. Here, we report that copper amine oxidases, which are important in metabolism of primary amines, catalyze a cyclic imine cleavage reaction. We isolate a microorganism (Arthrobacter sp. C-4A) which metabolizes a ß-carboline alkaloid, harmaline. The harmaline-metabolizing enzyme (HarA) purified from strain C-4A is found to be copper amine oxidase and catalyze a ring-opening reaction of cyclic imine within harmaline, besides oxidative deamination of amines. Growth experiments on strain C-4A and Western blot analysis indicate that the HarA expression is induced by harmaline. We propose a reaction mechanism of the cyclic imine cleavage by HarA containing a post-translationally-synthesized cofactor, topaquinone. Together with the above results, the finding of the same activity of copper amine oxidase from E. coli suggests that, in many living organisms, these enzymes may play crucial roles in metabolism of ubiquitous cyclic imines.

15.
Bioorg Med Chem ; 16(17): 8117-26, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18682327

RESUMO

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.


Assuntos
Dimetilaliltranstransferase/química , Flavonoides/síntese química , Macrolídeos/síntese química , Naftalenos/síntese química , Streptomyces/enzimologia , Catálise , Cristalografia por Raios X , Flavonoides/química , Cinética , Macrolídeos/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Naftalenos/química , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Tempo
16.
Sci Rep ; 8(1): 1282, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29352172

RESUMO

In the presence of CoA, cell-free extracts prepared from porcine liver was found to convert 7,8-dihydroxyflavone (DHF) to a pantetheine conjugate, which was a novel flavonoid. We purified a 7,8-DHF-converting enzyme from the extracts, and identified it as hemoglobin (Hb). The purified Hb showed the following two activities: (i) degradation of CoA into pantetheine through hydrolytic cleavage to yield pantetheine and 3'-phospho-adenosine-5'-diphosphate (ADP) independently of heme, and (ii) addition of a thiol (e.g., pantetheine, glutathione and cysteine) to 7,8-DHF through C-S bond formation. Human Hb also exhibited the above flavonoid-converting activity. In addition, heme-containing enzymes such as peroxidase and catalase added each of pantetheine, glutathione and cysteine to the flavonoid, although no pantetheine conjugates were synthesized when CoA was used as a substrate. These findings indicated that the thiol-conjugating activity is widely observed in heme-containing proteins. On the other hand, only Hb catalyzed the hydrolysis of CoA, followed by the thiol conjugation to synthesize the pantetheine conjugate. To the best of our knowledge, this is the first report showing that Hb has the catalytic ability to convert naturally occurring bioactive compounds, such as dietary flavonoids, to the corresponding conjugates in the presence of thiol donors or CoA.


Assuntos
Coenzima A/metabolismo , Flavonas/metabolismo , Hemoglobinas/metabolismo , Compostos de Sulfidrila/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Hidrólise , Fígado/metabolismo , Suínos
17.
J Antibiot (Tokyo) ; 70(4): 435-442, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27731335

RESUMO

The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for the selective substrate recognition and its activation (as an acyl-O-AMP intermediate) during ATP consumption. DhbE, a stand-alone adenylation domain, acts on an aromatic acid, 2,3-dihydroxybenzoic acid (DHB). This activation is the initial step of the synthesis of bacillibactin that is a high-affinity small-molecule iron chelator also termed siderophore. Subsequently, the activated DHB is transferred and attached covalently to a peptidyl carrier protein domain via a thioester bond. Adenylation domains belong to the superfamily of adenylate-forming enzymes including acetyl-CoA synthetase, acyl-CoA synthetase and firefly luciferase. We previously reported a novel N-acylation reaction for an acyl-CoA synthetase (AcsA) that originally catalyzes the formation of a thioester bond between an acid and CoA, yielding acyl-CoA. This novel reaction was also confirmed for acetyl-CoA synthetase and firefly luciferase, but not yet for an adenylation domain. Here, we for the first time demonstrated the synthesis of N-acyl-L-cysteine by a stand-alone adenylation domain, DhbE. When DHB and L-cysteine were used as substrates of DhbE, N-DHB-L-cysteine was formed. A Vmax value of 0.0156±0.0008 units mg-1 and Km values of 150±18.3 mM for L-cysteine and 0.0579±0.0260 mM for DHB were obtained in this novel reaction. Furthermore, DhbE synthesized N-benzoyl-L-cysteine when benzoic acid and L-cysteine were used as substrates. Through the N-acylation reaction of DhbE, we also succeeded in the synthesis of N-aromatic acyl compounds that have never previously been reported to be produced by this enzymatic method.


Assuntos
Adenina/química , Amidas/síntese química , Oligopeptídeos/biossíntese , Acetato-CoA Ligase/metabolismo , Acilação , Ácido Benzoico/metabolismo , Coenzima A Ligases/metabolismo , Cisteína/metabolismo , Escherichia coli/metabolismo , Cinética , Ligases/metabolismo , Luciferases/metabolismo , Especificidade por Substrato
18.
PLoS One ; 12(5): e0178974, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28558054

RESUMO

[This corrects the article DOI: 10.1371/journal.pone.0175846.].

19.
PLoS One ; 12(4): e0175846, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28410434

RESUMO

In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig's blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.


Assuntos
Proteínas de Bactérias/metabolismo , Hidroliases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Cromatografia Líquida de Alta Pressão , Guaiacol/química , Hidroliases/química , Hidroliases/genética , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Cinética , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Naftalenos/análise , Naftalenos/química , Naftalenos/metabolismo , Oxirredução , Pseudomonas chlororaphis/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato
20.
J Gen Appl Microbiol ; 62(4): 167-73, 2016 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-27250663

RESUMO

Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show ß-cyano-L-alanine (ß-CNAla) synthetic activity. ß-CNAla synthase is known to catalyze nitrile synthesis: the formation of ß-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and ß-chloro-DL-alanine. In addition, we purified ß-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5-9.0 at an optimal temperature of 40-50°C. The enzyme is specific for O-acetyl-L-serine and ß-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 µmol/min/mg.


Assuntos
Alanina/análogos & derivados , Cianetos/metabolismo , Liases/isolamento & purificação , Liases/metabolismo , Nitrilas/metabolismo , Pseudomonas/enzimologia , Alanina/biossíntese , Meios de Cultura/química , Cisteína/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Liases/biossíntese , Liases/química , Peso Molecular , Pseudomonas/metabolismo , Serina/metabolismo , Serina O-Acetiltransferase/metabolismo , Especificidade por Substrato , Temperatura
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