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1.
Org Biomol Chem ; 17(42): 9375-9389, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31621743

RESUMO

Squalene-hopene cyclase (SHC) converts acyclic squalene 1 into the 6,6,6,6,5-fused pentacyclic triterpenes hopene and hopanol. Previously, we reported the polycyclization products 14-17 of 27-norsqualene (13a) and 28-norsqualene (13b) by SHC, and suggested the importance of Me-27 of 1 for the normal polycyclization pathway. To further ensure the theory, (3R,S)-27-noroxidosqualenes (18 and 19) were incubated, and the structures of products 20-25 thus obtained prompted us to reinvestigate the SHC reaction of 13a (13b). One new product 29, composed of a 6,5 + 5,5 ring system with 13α-H and 17α-H, was obtained from 13a in addition to both the previously isolated products 14-17 and the 6,6,6,5-fused tetracyclic dammarenyl compounds, which were overlooked before. We propose the name "nor-allodammarane" for this novel tetracyclic 6,5 + 5,5 ring system and the name "nor-allogammacerane" for the pentacyclic 6,5 + 5,5 + 6 ring system. The stereochemistry of 29 indicated that 13a folded in the following chair-boat-boat-boat conformation: 10α-H, 11ß-H; 14α-H, 15ß-Me; 18α-H and 19ß-Me, which further allowed us to predict the configuration of 20R for 14 and that of 20S for 15. Substrates 18 and 19 were also cyclized only into allodammarane scaffolds 20-25, and all the structures of 20-25 further indicated that 18 and 19 also folded in the same conformation as 13a, providing further evidence that Me-27 groups of 1 and oxidosqualene are essential for the normal polycyclization pathway by SHC.

2.
Chembiochem ; 19(5): 486-495, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29214725

RESUMO

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli ß-amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild-type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π-electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation-π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E-ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56-78 % of wild-type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH-π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation-π interaction.


Assuntos
Euphorbia/enzimologia , Transferases Intramoleculares/metabolismo , Vias Biossintéticas , Cátions/química , Cátions/metabolismo , Ciclização , Estabilidade Enzimática , Euphorbia/química , Euphorbia/genética , Euphorbia/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Modelos Moleculares , Mutagênese , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Mutação Puntual , Conformação Proteica , Triptofano/química , Triptofano/genética , Triptofano/metabolismo
3.
Nat Prod Commun ; 11(2): 163-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27032191

RESUMO

The enzymatic reactions of geranylfarnesol (8) and its acetate 9, classified as sesterterpenes (C25), using squalene-hopene cyclase (SHC) were investigated. The enzymatic reaction of 8 afforded 6/6-fused bicyclic 20, 6/6/6-fused tricyclic 21, and 6/6/6/6-fused tetracyclic compounds 22 and 23 as the main products (35% yield), whereas that of 9 afforded two 6/6/6-fused tricyclic compounds 24 and 25 in a high yield (76.3%) and a small amount (5.0%) of 26 (the acetate of 22). A significantly higher conversion of 9 indicates that the arrangement of the substrate in the reaction cavity changed. The lipophilic nature and/or the bulkiness of the acetyl group may have changed its binding with SHC, thus placing the terminal double bond of 9 in the vicinity of the DXDD motif of SHC, which is responsible for the proton attack on the double bond to initiate the polycyclization reaction. The results obtained for 8 are different to some extent than those reported by Shinozaki et al. The products obtained in this study were deprotonated compounds; however, the products reported by Shinozaki et al. were hydroxylated compounds.


Assuntos
Alicyclobacillus/enzimologia , Proteínas de Bactérias/metabolismo , Gefarnato/análogos & derivados , Regulação Bacteriana da Expressão Gênica/fisiologia , Transferases Intramoleculares/metabolismo , Proteínas de Bactérias/química , Gefarnato/química , Regulação Enzimológica da Expressão Gênica , Transferases Intramoleculares/química , Estrutura Molecular
4.
Chembiochem ; 16(5): 772-81, 2015 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-25694050

RESUMO

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)-kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)-O-methylkolavelool from (+)-kolavelool, so this enzyme was an O-methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)-O-methylkolavelool biosynthetic gene cluster.


Assuntos
Chloroflexi/genética , Chloroflexi/metabolismo , Diterpenos/metabolismo , Família Multigênica , Chloroflexi/citologia , Diterpenos/química , Metiltransferases/genética , Metiltransferases/metabolismo , Conformação Molecular
5.
Sci Rep ; 4: 6214, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25269599

RESUMO

We report the first X-ray crystal structure of ent-kaur-16-ene synthase from Bradyrhizobium japonicum, together with the results of a site-directed mutagenesis investigation into catalytic activity. The structure is very similar to that of the α domains of modern plant terpene cyclases, a result that is of interest since it has been proposed that many plant terpene cyclases may have arisen from bacterial diterpene cyclases. The ent-copalyl diphosphate substrate binds to a hydrophobic pocket near a cluster of Asp and Arg residues that are essential for catalysis, with the carbocations formed on ionization being protected by Leu, Tyr and Phe residues. A bisphosphonate inhibitor binds to the same site. In the kaurene synthase from the moss Physcomitrella patens, 16-α-hydroxy-ent-kaurane as well as kaurene are produced since Leu and Tyr in the P. patens kaurene synthase active site are replaced by smaller residues enabling carbocation quenching by water. Overall, the results represent the first structure determination of a bacterial diterpene cyclase, providing insights into catalytic activity, as well as structural comparisons with diverse terpene synthases and cyclases which clearly separate the terpene cyclases from other terpene synthases having highly α-helical structures.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Bradyrhizobium/enzimologia , Diterpenos do Tipo Caurano/metabolismo , Alquil e Aril Transferases/genética , Cromatografia em Gel , Clonagem Molecular , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Especificidade por Substrato
6.
FEBS J ; 281(24): 5479-97, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25283713

RESUMO

Information regarding squalene synthases (SQSs) from prokaryotes is scarce. We aimed to characterize the SQS from Methylococcus capsulatus. We studied its reaction mechanism by kinetic analysis and evaluated the structure of the substrate/inhibitor-binding sites via homology modeling. The cloned M. capsulatus SQS was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid column chromatography. Interestingly, M. capsulatus SQS was water-soluble and did not require any detergent for its higher activity, unlike other SQSs studied previously; supplementation of any type of detergent inhibited enzyme activity. The specific activity and the kinetic values (Km and kcat ) for the substrate farnesyl diphosphate and NADPH are reported. The substrate analog farnesyl methylenediphosphonate showed potent inhibition toward the enzyme. We prepared the site-specific mutants directed at potential active-site residues (58) DXX(61) E(62) D (S1 site) and (213) DXX(216) D(217) D (S2 site), which were assumed to be involved in the binding of the substrate farnesyl diphosphate through the Mg(2+) ion. We first demonstrated that the S1 site and the two basic residues (R55 and K212) were responsible for the binding of farnesyl diphosphate. Furthermore, we examined the catalytic roles of the highly conserved aromatic residues and demonstrated that the Y164 residue abstracts the proton of cation 5, which is produced during the first half-reaction (Scheme 1), to afford presqualene diphosphate, and that the W224 residue stabilizes the intermediary cation 5 via the cation-π interaction. Furthermore, we confirm for the first time that the F32 and the Y51 residues also stabilize the carbocation intermediate(s) generated during the second half-reaction.


Assuntos
Motivos de Aminoácidos , Sequência Conservada , Farnesil-Difosfato Farnesiltransferase/metabolismo , Methylococcus capsulatus/enzimologia , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Farnesil-Difosfato Farnesiltransferase/química , Cinética , Mutagênese Sítio-Dirigida
7.
Org Biomol Chem ; 12(23): 3836-46, 2014 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-24695673

RESUMO

ß-Amyrin, a triterpene, is widely distributed in plants and its glycosides confer important biological activities. Mutagenesis studies on ß-amyrin synthase are very limited as compared with those of squalene-hopene cyclase and lanosterol synthase. This study was conducted to elucidate the function of the F474 residue of Euphorbia tirucalli ß-amyrin cyclase, which is highly conserved in the superfamily of oxidosqualene cyclases. Nine site-specific variants with Gly, Ala, Val, Leu, Met, Tyr, Trp, His, and Thr were constructed. We isolated 9 products from these mutants in addition to ß-amyrin and determined the chemical structures. The Gly and Ala mutants produced significantly larger amounts of the bicyclic products and a decreased amount of ß-amyrin, indicating that the F474 residue was located near the B-ring formation site. Surprisingly, the Ala variant produced (9ßH)-polypoda-7,13,17,21-tetraen-3ß-ol and (9ßH)-polypoda-8(26),13,17,21-tetraen-3ß-ol, which are generated from a chair-boat folding conformation. This is the first report describing the conformational change from the chair-chair into the chair-boat folding conformation among the reported mutagenesis studies of oxidosqualene cyclases. Substitution with aliphatic amino acids lacking π-electrons such as Val, Leu, and Met led to a significantly decreased production of bicyclic compounds, and in turn exhibited a higher production of ß-amyrin. Furthermore, the Leu and Met variants exhibited high enzymatic activities: ca. 74% for Leu and ca. 91% for Met variants as compared to the wild-type. These facts unambiguously demonstrate that the major role of Phe474 is not to stabilize the transient cation via cation-π interaction, but is to confer the appropriate steric bulk near the B-ring formation site, leading to the completion of the normal polycyclization pathway without accumulation of abortive cyclization products.


Assuntos
Elétrons , Euphorbia/enzimologia , Transferases Intramoleculares/metabolismo , Ácido Oleanólico/análogos & derivados , Fenilalanina/química , Cromatografia Gasosa , Ciclização , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Ácido Oleanólico/química , Ácido Oleanólico/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
8.
J Am Chem Soc ; 136(7): 2892-6, 2014 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-24475925

RESUMO

We have obtained the structure of the bacterial diterpene synthase, tuberculosinol/iso-tuberculosinol synthase (Rv3378c) from Mycobacterium tuberculosis , a target for anti-infective therapies that block virulence factor formation. This phosphatase adopts the same fold as found in the Z- or cis-prenyltransferases. We also obtained structures containing the tuberculosinyl diphosphate substrate together with one bisphosphonate inhibitor-bound structure. These structures together with the results of site-directed mutagenesis suggest an unusual mechanism of action involving two Tyr residues. Given the similarity in local and global structure between Rv3378c and the M. tuberculosis cis-decaprenyl diphosphate synthase (DPPS; Rv2361c), the possibility exists for the development of inhibitors that target not only virulence but also cell wall biosynthesis, based in part on the structures reported here.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/química , Diterpenos/metabolismo , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Difosfonatos/química , Difosfonatos/metabolismo , Difosfonatos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
9.
Chembiochem ; 14(7): 822-5, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23554321

RESUMO

Basic enzyme: The tetraprenyl-ß-curcumene synthase homologue from the alkalophilic Bacillus clausii catalyses conversions of a geranylfarnesyl diphosphate and a hexaprenyl diphosphate into novel head-to-tail acyclic sesterterpene and triterpene. Tetraprenyl-ß-curcumene synthase homologues represent a new family of terpene synthases that form not only sesquarterpene but also sesterterpene and triterpene.


Assuntos
Alquil e Aril Transferases/isolamento & purificação , Alquil e Aril Transferases/metabolismo , Bacillus/enzimologia , Terpenos/química , Terpenos/metabolismo , Alquil e Aril Transferases/classificação , Espectroscopia de Ressonância Magnética , Especificidade por Substrato
10.
J Antibiot (Tokyo) ; 65(11): 551-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22872183

RESUMO

Recent bacterial genome sequencing projects have shown the presence of many putative sesquiterpene cyclase (SC) genes, especially in the Gram-positive, filamentous bacterial genus Streptomyces. We describe here the characterization of a SC gene (SGR6065, named gecA) from Streptomyces griseus. Overexpression of gecA in Streptomyces lividans produced a sesquiterpene, which was isolated and determined to be (+)-epicubenol using spectroscopic analyses. The N-terminal histidine-tagged GecA protein was produced in Escherichia coli. Incubation of the recombinant GecA protein with farnesyl diphosphate (FPP) yielded (+)-epicubenol as the major product. The K(m) value for FPP and the k(cat) value for (+)-epicubenol formation were calculated to be 254 ± 7.1 nM and 0.026 ± 0.001 s(-1), respectively. The k(cat)/K(m) value (0.10 s(-1) µM(-1)) was broadly comparable to those reported for known bacterial SCs. (+)-Epicubenol was detected in the crude cell lysate of wild-type S. griseus, but not in a gecA-knockout mutant, indicating that GecA is a genuine (+)-epicubenol synthase. Although (+)-epicubenol synthases have been previously purified and characterized from the liverwort Heteroscyphus planus and Streptomyces sp. LL-B7, no (+)-epicubenol synthase gene has been cloned to date. The gecA gene is thus the first example of an (+)-epicubenol synthase-encoding gene. (+)-Epicubenol production was not controlled by the microbial hormone A-factor that induces morphological differentiation and production of several secondary metabolites in S. griseus.


Assuntos
Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Sesquiterpenos/metabolismo , Streptomyces griseus/enzimologia , Streptomyces griseus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , Cromatografia Gasosa-Espectrometria de Massas , Técnicas de Inativação de Genes , Genes Bacterianos , Isomerases/genética , Isomerases/metabolismo , Cinética , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sesquiterpenos/química , Estereoisomerismo , Transcriptoma
11.
J Bacteriol ; 194(6): 1544-51, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22247507

RESUMO

Streptomyces griseus contains the srs operon, which is required for phenolic lipid biosynthesis. The operon consists of srsA, srsB, and srsC, which encode a type III polyketide synthase, an O-methyltransferase, and a flavoprotein hydroxylase, respectively. We previously reported that the recombinant SrsA protein synthesized 3-(13'-methyltetradecyl)-4-methylresorcinol, using iso-C(16) fatty acyl-coenzyme A (CoA) as a starter substrate and malonyl-CoA and methylmalonyl-CoA as extender substrates. An in vitro SrsA reaction using [(13)C(3)]malonyl-CoA confirmed that the order of extender substrate condensation was methylmalonyl-CoA, followed by two extensions with malonyl-CoA. Furthermore, SrsA was revealed to produce an alkylresorcylic acid as its direct product rather than an alkylresorcinol. The functional SrsB protein was produced in the membrane fraction in Streptomyces lividans and used for the in vitro SrsB reaction. When the SrsA reaction was coupled, SrsB produced alkylresorcinol methyl ether in the presence of S-adenosyl-l-methionine (SAM). SrsB was incapable of catalyzing the O-methylation of alkylresorcinol, indicating that alkylresorcylic acid was the substrate of SrsB and that SrsB catalyzed the conversion of alkylresorcylic acid to alkylresorcinol methyl ether, namely, by both the O-methylation of the hydroxyl group (C-6) and the decarboxylation of the neighboring carboxyl group (C-1). O-methylated alkylresorcylic acid was not detected in the in vitro SrsAB reaction, although it was presumably stable, indicating that O-methylation did not precede decarboxylation. We therefore postulated that O-methylation was coupled with decarboxylation and proposed that SrsB catalyzed the feasible SAM-dependent decarboxylative methylation of alkylresorcylic acid. To the best of our knowledge, this is the first report of a methyltransferase that catalyzes decarboxylative methylation.


Assuntos
Hidroxibenzoatos/metabolismo , Metiltransferases/metabolismo , Streptomyces griseus/enzimologia , Streptomyces griseus/metabolismo , Metabolismo dos Lipídeos , Metilação , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Fenóis/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces lividans/genética , Especificidade por Substrato
14.
J Biol Chem ; 286(32): 27980-7, 2011 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-21693706

RESUMO

Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-ß-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-ß-caryophyllene. Several ß-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.


Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Carbono Liases/metabolismo , Sesquiterpenos/metabolismo , Streptomyces griseus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono-Carbono Liases/química , Carbono-Carbono Liases/genética , Escherichia coli/genética , Sesquiterpenos Policíclicos , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sesquiterpenos/química , Streptomyces griseus/genética
15.
Org Lett ; 13(10): 2678-81, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21526825

RESUMO

The gene products of AK121211, AK066327, and AK070534 from Oryza sativa encode cycloartenol, parkeol, and achilleol B synthases, respectively. Parkeol synthase is a unique enzyme that affords parkeol as a single product. Achilleol B synthase is the third seco-type triterpene cyclase identified to date, and triterpenes produced by this synthase include achilleol B (90%), tetracyclic (5.12%) and pentacyclic scaffolds (4.37%), and unidentified triterpenes (0.51%). The pathway for achilleol B biosynthesis is proposed.


Assuntos
Liases Intramoleculares/metabolismo , Transferases Intramoleculares/metabolismo , Oryza/enzimologia , Triterpenos/isolamento & purificação , Transferases Intramoleculares/genética , Transferases Intramoleculares/isolamento & purificação , Lanosterol/análogos & derivados , Lanosterol/química , Lanosterol/isolamento & purificação , Lanosterol/metabolismo , Estrutura Molecular , Oryza/genética , Fitosteróis/química , Fitosteróis/isolamento & purificação , Fitosteróis/metabolismo , Triterpenos/química , Triterpenos/metabolismo
16.
Org Biomol Chem ; 9(7): 2156-65, 2011 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-21290071

RESUMO

The Rv3378c gene product from Mycobacterium tuberculosis encodes a diterpene synthase to produce tuberculosinol (3), 13R-isotuberculosinol (4a), and 13S-isotuberculosinol (4b) from tuberculosinyl diphosphate (2). The product distribution ratios are 1 : 1 for 3 to 4 and 1 : 3 for 4a to 4b. The substrate specificity of the Rv3378c-encoded enzyme was examined. The 3 labdadienyl diphosphates, copalyl diphosphate (CDP) (7), ent-CDP (8), and syn-CDP (9), underwent the conversion reaction, with good yields (67-78%). Copalol (23) and manool (24) were produced from 7, ent-copalol (25) and ent-manool (26) from 8, and syn-copalol (27) and vitexifolin A (28) from 9. The ratio of 23 to 24 was 40 : 27, that of 25:26 was 22 : 50, and that of 27:28 was 16 : 62. Analysis on a GC-MS chromatograph equipped with a chiral column revealed that 24, 26, and 28 consisted of a mixture of 13R- (a) and 13S-stereoisomers (b) in the following ratio: ca. 1 : 1 for 24a to 24b, ca. 1 : 5 for 26a to 26b, and ca. 1 : 19 for 28a to 28b. The structures of these products indicate that the reactions of the 3 CDPs proceeded in the same fashion as that of 2. This is the first report on the enzymatic synthesis of natural diterpenes manool, ent-manool, and vitexifolin A. Both Rv3377c and Rv3378c genes are found in virulent Mycobacterium species, but not in avirulent species. We found that 3 and 4 inhibited the phagocytosis of opsonized zymosan particles by human macrophage-like cells. Interestingly, the inhibitory activity was synergistically increased by the coexistence of 3 and 4b. Other labdane-related diterpenes, 13-16 and 23-28, had little or no inhibitory activity. This synergistic inhibition by 3 and 4 may provide further advantage to the impairment of phagocyte function, which might contribute to pathogenicity of M. tuberculosis.


Assuntos
Compostos Bicíclicos com Pontes/química , Proteínas de Ligação a DNA/metabolismo , Diterpenos/química , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Fagocitose/efeitos dos fármacos , Biocatálise , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Diterpenos/farmacologia , Humanos , Estrutura Molecular , Especificidade por Substrato
17.
Biosci Biotechnol Biochem ; 75(1): 75-81, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21228491

RESUMO

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of ¹H- and ¹³C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.


Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Diterpenos/metabolismo , Mycobacterium tuberculosis/genética , Alquil e Aril Transferases/química , Alquil e Aril Transferases/isolamento & purificação , Motivos de Aminoácidos , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Genoma Bacteriano/genética , Cinética , Espectroscopia de Ressonância Magnética , Metais/farmacologia , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/enzimologia , Multimerização Proteica , Estrutura Quaternária de Proteína
18.
Chembiochem ; 12(15): 2271-5, 2011 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-23106076

RESUMO

Now found in bacteria: An increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. The characterization of two sesquiterpene cyclase (SC) genes found in the draft genome sequence of Streptomyces citricolor is described here. Our study strongly supports the idea that genome mining is a useful approach in revealing the terpenoid diversity in bacteria.


Assuntos
Carbono-Carbono Liases/metabolismo , Sesquiterpenos de Germacrano/metabolismo , Sesquiterpenos/metabolismo , Streptomyces/enzimologia , Carbono-Carbono Liases/genética , Genes Bacterianos , Sesquiterpenos Monocíclicos , Sesquiterpenos/química , Sesquiterpenos de Germacrano/química , Streptomyces/genética
19.
Chembiochem ; 10(12): 2060-71, 2009 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-19618417

RESUMO

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 degrees C, Mg(II) (0.1 mM), and Triton X-100 (0.1 %). Under these conditions, the kinetic values of K(M) and k(cat) were determined to be 11.7+/-1.9 microM for GGPP and 12.7+/-0.7 min(-1), respectively, whereas the specific activity was 186 nmol min(-1) mg(-1). The enzyme activity was inhibited at substrate concentrations higher than 50 microM. The catalytic activity was strongly inhibited by 15-aza-dihydrogeranylgeraniol and 5-isopropyl-N,N,N,2-tetramethyl-4-(piperidine-1-carbonyloxy)benzenaminium chloride (Amo-1618). The DXDTT(293-297) motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen-bonding networks. The Rv3377c enzyme could also react with (14R/S)-14,15-oxidoGGPP to generate 3alpha- and 3beta-hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium-labeled GGPP and oxidoGGPP. We found that GGPP and (14R)-oxidoGGPP adopted a chair/chair conformation, but (14S)-oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A-ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)-Oxidosqualene is folded in a boat conformation, whereas (3S)-2,3-oxidosqualene folds into a chair conformation, for the formation of the A-rings of the hopene and tetrahymanol skeletons, respectively.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Diterpenos/metabolismo , Genoma Bacteriano , Liases/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Ciclização , Diterpenos/farmacologia , Liases/antagonistas & inibidores , Liases/química , Liases/genética , Dados de Sequência Molecular , Estrutura Molecular , Fosfatos de Poli-Isoprenil/síntese química , Fosfatos de Poli-Isoprenil/metabolismo , Compostos de Amônio Quaternário/farmacologia
20.
J Bacteriol ; 191(15): 4916-23, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19465653

RESUMO

Type III polyketide synthases (PKSs) synthesize a variety of aromatic polyketides in plants, fungi, and bacteria. The bacterial genome projects predicted that probable type III PKS genes are distributed in a wide variety of gram-positive and -negative bacteria. The gram-positive model microorganism Bacillus subtilis contained the bcsA-ypbQ operon, which appeared to encode a type III PKS and a methyltransferase, respectively. Here, we report the characterization of bcsA (renamed bpsA, for Bacillus pyrone synthase, on the basis of its function) and ypbQ, which are involved in the biosynthesis of aliphatic polyketides. In vivo analysis demonstrated that BpsA was a type III PKS catalyzing the synthesis of triketide pyrones from long-chain fatty acyl-coenzyme A (CoA) thioesters as starter substrates and malonyl-CoA as an extender substrate, and YpbQ was a methyltransferase acting on the triketide pyrones to yield alkylpyrone methyl ethers. YpbQ thus was named BpsB because of its functional relatedness to BpsA. In vitro analysis with histidine-tagged BpsA revealed that it used broad starter substrates and produced not only triketide pyrones but also tetraketide pyrones and alkylresorcinols. Although the aliphatic polyketides were expected to localize in the membrane and play some role in modulating the rigidity and properties of the membrane, no detectable phenotypic changes were observed for a B. subtilis mutant containing a whole deletion of the bpsA-bpsB operon.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Macrolídeos/metabolismo , Metiltransferases/metabolismo , Policetídeo Sintases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cinética , Lipídeos/análise , Lipídeos/química , Metiltransferases/genética , Óperon/genética , Policetídeo Sintases/genética , Especificidade por Substrato
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