RESUMO
The published online version contains mistake in the chemical structure of scyllo-inosose in Fig. 5 and Fig. 7. The correct configuration of 1-hydroxyl group in scyllo-inosose should have been the same to myo-inositol.
RESUMO
(-)-vibo-Quercitol is a deoxyinositol (1L-1,2,4/3,5-cyclohexanepentol) that occurs naturally in low concentrations in oak species, honeydew honey, and Gymnema sylvestre. The author's research group recently reported that (-)-vibo-quercitol and scyllo-quercitol (2-deoxy-myo-inositol, 1,3,5/2,4-cyclohexanepentol), a stereoisomer of (-)-vibo-quercitol, are stereoselectively synthesized from 2-deoxy-scyllo-inosose by the reductive reaction of a novel (-)-vibo-quercitol 1-dehydrogenase in Burkholderia terrae and of a known scyllo-inositol dehydrogenase in Bacillus subtilis, respectively. The author's research group therefore identified two enzymes capable of producing both stereoisomers of deoxyinositols, which are rare in nature. (-)-vibo-Quercitol and scyllo-quercitol are potential intermediates for pharmaceuticals. In this review, the author describes the biosynthesis and enzymatic production of quercitols and myo-inositol stereoisomers and their application in the production of potential pharmaceuticals.
Assuntos
Bacillus subtilis/enzimologia , Burkholderiaceae/enzimologia , Indústria Farmacêutica/métodos , Inositol/biossíntese , Preparações Farmacêuticas/síntese química , Inositol/química , Inositol/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , EstereoisomerismoRESUMO
(-)-vibo-Quercitol is a deoxyinositol (1L-1,2,4/3,5-cyclohexanepentol) that naturally occurs in oak species, honeydew honey, wines aged in oak barrels, and Gymnema sylvestre and is a potential intermediate for pharmaceuticals. We found that (-)-vibo-quercitol is stereoselectively synthesized from 2-deoxy-scyllo-inosose by the reductive reaction of a novel (-)-vibo-quercitol 1-dehydrogenase found in the proteomes of Burkholderia, Pseudomonas, and Arthrobacter. Among them, Burkholderia terrae sp. AKC-020 (40-1) produced a (-)-vibo-quercitol 1-dehydrogenase appropriate for synthesizing (-)-vibo-quercitol with a high diastereomeric excess. The enzyme was strongly induced in Bu. terrae cells when quercitol or 2-deoxy-scyllo-inosose was used as carbon source in the culture medium. The enzyme is NAD(H)-dependent and shows highly specific activity for (-)-vibo-quercitol and myo-inositol among the substrates tested. The enzyme gene (qudh) was obtained by PCR using degenerate primers based on the N-terminal and internal amino acid sequences of the purified enzyme, followed by thermal asymmetric interlaced PCR. The qudh gene showed homology with inositol 2-dehydrogenase (sharing 49.5% amino acid identity with IdhA from Sinorhizobium meliloti 1021). We successfully produced several recombinant (-)-vibo-quercitol 1-dehydrogenases and related enzymes identified by genome database mining using an Escherichia coli expression system. This revealed that scyllo-inositol dehydrogenase (IolX) in Bacillus subtilis can catalyze the reduction of 2-deoxy-scyllo-inosose to yield scyllo-quercitol, a stereoisomer of (-)-vibo-quercitol. Thus, we successfully identified two enzymes to produce both stereoisomers of deoxyinositols that are rare in nature.
Assuntos
Burkholderiaceae/enzimologia , Inositol/análogos & derivados , Oxirredutases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Burkholderiaceae/genética , Clonagem Molecular , Coenzimas/metabolismo , Meios de Cultura/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Inositol/metabolismo , NAD/metabolismo , Oxirredutases/química , Oxirredutases/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
BACKGROUND: Flavonoids are secondary metabolites that play significant roles in plant cells. In particular, polymethoxy flavonoids (PMFs), including nobiletin, have been reported to exhibit various health-supporting properties such as anticancer, anti-inflammatory, and anti-pathogenic properties. However, it is difficult to utilize PMFs for medicinal and dietary use because plant cells contain small amounts of these compounds. Biosynthesis of PMFs in plant cells is carried out by the methylation of hydroxyl groups of flavonoids by O-methyltransferases (FOMT), and many kinds of FOMTs with different levels of substrate specificity and regioselectivity are cooperatively involved in this biosynthesis. RESULTS: In this study, we isolated five genes encoding FOMT (CdFOMT1, 3, 4, 5, and 6) from Citrus depressa, which is known to accumulate nobiletin in the peels of its fruits. The genes encoded Mg(2+)-independent O-methyltransferases and showed high amino acid sequence similarity (60-95 %) with higher plant flavonoid O-methyltransferases. One of these genes is CdFOMT5, which was successfully expressed as a soluble homodimer enzyme in Escherichia coli. The molecular mass of the recombinant CdFOMT5 subunit was 42.0 kDa including a 6× histidine tag. The enzyme exhibited O-methyltransferase activity for quercetin, naringenin, (-)-epicatechin, and equol using S-adenosyl-L-methionine (SAM) as a methyl donor, and its optimal pH and temperature were pH 7.0 and 45 °C, respectively. The recombinant CdFOMT5 demonstrated methylation activity for the 3-, 5-, 6-, and 7-hydroxyl groups of flavones, and 3,3',5,7-tetra-O-methylated quercetin was synthesized from quercetin as a final product of the whole cell reaction system. Thus, CdFOMT5 is a O-methyltransferase possessing a broad range of substrate specificity and regioselectivity for flavonoids. CONCLUSIONS: Five FOMT genes were isolated from C. depressa, and their nucleotide sequences were determined. CdFOMT5 was successfully expressed in E. coli cells, and the enzymatic properties of the recombinant protein were characterized. Recombinant CdFOMT5 indicated O-methyltransferase activity for many flavonoids and a broad regioselectivity for quercetin as a substrate. Whole-cell biocatalysis using CdFOMT5 expressed in E. coli cells was performed using quercetin as a substrate, and 3,3',5,7-tetramethylated quercetin was obtained as the final product.
Assuntos
Citrus/enzimologia , Clonagem Molecular , Metiltransferases/química , Metiltransferases/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sequência de Aminoácidos , Citrus/química , Citrus/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Flavonoides/metabolismo , Metilação , Metiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
We describe the development of biocatalysis for producing optically pure straight-chain (S)-epoxyalkanes using styrene monooxygenase of Rhodococcus sp. strain ST-10 (RhSMO). RhSMO was expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, and the bioconversion reaction was performed in an organic solvent-water biphasic reaction system. The biocatalytic process enantioselectively converted linear terminal alkenes to their corresponding (S)-epoxyalkanes using glucose and molecular oxygen. When 1-heptene and 6-chloro-1-hexene were used as substrates (400 mM) under optimized conditions, 88.3 mM (S)-1,2-epoxyheptane and 246.5 mM (S)-1,2-epoxy-6-chlorohexane, respectively, accumulated in the organic phase with good enantiomeric excess (ee; 84.2 and 95.5%). The biocatalysis showed broad substrate specificity toward various aliphatic alkenes, including functionalized and unfunctionalized alkenes, with good to excellent ee. Here, we demonstrate that this biocatalytic system is environmentally friendly and useful for producing various enantiopure (S)-epoxyalkanes.
Assuntos
Alcanos/metabolismo , Micrococcus luteus/enzimologia , Micrococcus luteus/metabolismo , Oxigenases/metabolismo , Rhodococcus/enzimologia , Biotransformação , Expressão Gênica , Glucose/metabolismo , Micrococcus luteus/genética , Oxigênio/metabolismo , Oxigenases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodococcus/genética , Especificidade por SubstratoRESUMO
Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (â¼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols.
Assuntos
Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Metagenoma , Actinomycetales/genética , Álcoois/metabolismo , Sequência de Aminoácidos , Biotecnologia/métodos , Catálise , Clonagem Molecular , Primers do DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Biblioteca Gênica , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The asymmetric reduction of ketones is one of the most promising processes for producing chiral alcohols. However, dehydrogenases or reductases that can catalyze the reduction of ketones to give anti-Prelog chiral alcohols have been limited to some NADP(+)/NADPH-dependent enzymes. Recently, we reported a novel NAD(+)/NADH-dependent alcohol dehydrogenase (ADH) from Leifsonia sp. and Pseudomonas ADH homologs from soil metagenomes. Moreover, we have established an efficient hydrogen-transfer bioreduction process with 2-propanol as a hydrogen donor using Leifsonia ADH. This review focuses on the recent development of novel ADHs for producing industrially useful anti-Prelog chiral alcohols from various ketones.
Assuntos
Actinobacteria/enzimologia , Álcool Desidrogenase/metabolismo , Álcoois/metabolismo , Pseudomonas/enzimologia , 2-Propanol/metabolismo , Biotecnologia/métodos , Biotecnologia/tendências , Hidrogênio/metabolismo , Cetonas/metabolismo , OxirreduçãoRESUMO
Chiral alcohols are valuable as diverse chemicals and synthetic intermediate materials. Phenylacetaldehyde reductase (PAR) is an enzyme that converts a wide variety of ketones into chiral alcohols with high optical purity. When an alcohol such as 2-propanol is used as a hydrogen donor, PAR itself will also mediate the regeneration of the coenzyme NADH in situ. Perceiving a capacity for improvement, we sought to develop a PAR that is able to convert higher concentrations of substrates in the presence of high concentrations of 2-propanol. The selection procedure for mutants was re-examined and a procedure able to select an effective amino acid substitution was established. Two advantageous amino acid substitutions were successfully selected using the procedure. When high-concentration substrate conversion reaction was subjected with a mutant that integrated both the two amino acid substitutions, near-complete conversions of m-chlorophenacyl chloride (m-CPC) (2.1 mmol/ml) and ethyl 4-chloro-3-oxobutanoate (ECOB) (1.9 mmol/ml) were achieved.
Assuntos
2-Propanol/metabolismo , Oxirredutases do Álcool/metabolismo , Rhodococcus/enzimologia , Seleção Genética , Oxirredutases do Álcool/genética , Substituição de Aminoácidos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Rhodococcus/genéticaRESUMO
We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(-)-3-quinuclidinol.
Assuntos
Actinomycetales/enzimologia , Coenzimas/metabolismo , NAD/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Quinuclidinas/metabolismo , Actinomycetales/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced (S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding (S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Compostos de Epóxi/metabolismo , Expressão Gênica , Oxigenases/química , Oxigenases/metabolismo , Rhodococcus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Estabilidade Enzimática , Compostos de Epóxi/química , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Oxigenases/genética , Rhodococcus/química , Rhodococcus/genética , Alinhamento de Sequência , Estereoisomerismo , Estireno/metabolismoRESUMO
We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K(m) and V(max) values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium.
Assuntos
2-Propanol , Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/metabolismo , Álcoois/metabolismo , Meios de Cultura/química , Engenharia Genética/métodos , 2-Propanol/análise , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Álcoois/química , Biocatálise , Biotecnologia/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrogênio/metabolismo , Modelos Moleculares , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato , Água/análiseRESUMO
We found two NADH-dependent reductases (QNR and bacC) in Microbacterium luteolum JCM 9174 (M. luteolum JCM 9174) that can reduce 3-quinuclidinone to optically pure (R)-(-)-3-quinuclidinol. Alcohol dehydrogenase from Leifsonia sp. (LSADH) was combined with these reductases to regenerate NAD+ to NADH in situ in the presence of 2-propanol as a hydrogen donor. The reductase and LSADH genes were efficiently expressed in E. coli cells. A number of constructed E. coli biocatalysts (intact or immobilized) were applied to the resting cell reaction and optimized. Under the optimized conditions, (R)-(-)-3-quinuclidinol was synthesized from 3-quinuclidinone (15% w/v, 939 mM) giving a conversion yield of 100% for immobilized QNR. The optical purity of the (R)-(-)-3-quinuclidinol produced by the enzymatic reactions was >99.9%. Thus, E. coli biocatalysis should be useful for the practical production of the pharmaceutically important intermediate, (R)-(-)-3-quinuclidinol.
Assuntos
Actinomycetales/enzimologia , Álcool Desidrogenase/metabolismo , Escherichia coli/metabolismo , NADH NADPH Oxirredutases/metabolismo , Quinuclidinas/metabolismo , Álcool Desidrogenase/genética , Biocatálise , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , NADH NADPH Oxirredutases/genética , Rotação Ocular , Quinuclidinas/química , Estereoisomerismo , Fatores de TempoRESUMO
We recently demonstrated that the futalosine pathway was operating in some bacteria for the biosynthesis of menaquinone and that futalosine was converted into dehypoxanthinyl futalosine (DHFL) by an MqnB of Thermus thermophilus. In this study, we found that aminodeoxyfutalosine, which has adenine instead of hypoxanthine in futalosine, was directly converted into DHFL by an MqnB of Helicobacter pylori. Therefore, this step is potentially an attractive target for the development of specific anti-H. pylori drugs.
Assuntos
Bactérias/metabolismo , Helicobacter pylori/metabolismo , Hidrolases/metabolismo , Nucleosídeos/metabolismo , Adenina/química , Bactérias/enzimologia , Cromatografia Líquida de Alta Pressão , Helicobacter pylori/enzimologia , Helicobacter pylori/genética , Hidrolases/genética , Hipoxantina/metabolismo , Nucleosídeos/química , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo , Vitamina K 2/metabolismoRESUMO
Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.
RESUMO
BACKGROUND: Biogenic emissions of methyl halides (CH3Cl, CH3Br and CH3I) are the major source of these compounds in the atmosphere; however, there are few reports about the halide profiles and strengths of these emissions. Halide ion methyltransferase (HMT) and halide/thiol methyltransferase (HTMT) enzymes concerning these emissions have been purified and characterized from several organisms including marine algae, fungi, and higher plants; however, the correlation between emission profiles of methyl halides and the enzymatic properties of HMT/HTMT, and their role in vivo remains unclear. RESULTS: Thirty-five higher plant species were screened, and high CH3I emissions and HMT/HTMT activities were found in higher plants belonging to the Poaceae family, including wheat (Triticum aestivum L.) and paddy rice (Oryza sativa L.), as well as the Brassicaceae family, including daikon radish (Raphanus sativus). The in vivo emission of CH3I clearly correlated with HMT/HTMT activity. The emission of CH3I from the sprouting leaves of R. sativus, T. aestivum and O. sativa grown hydroponically increased with increasing concentrations of supplied iodide. A gene encoding an S-adenosylmethionine halide/thiol methyltransferase (HTMT) was cloned from R. sativus and expressed in Escherichia coli as a soluble protein. The recombinant R. sativus HTMT (RsHTMT) was revealed to possess high specificity for iodide (I-), bisulfide ([SH]-), and thiocyanate ([SCN]-) ions. CONCLUSION: The present findings suggest that HMT/HTMT activity is present in several families of higher plants including Poaceae and Brassicaceae, and is involved in the formation of methyl halides. Moreover, it was found that the emission of methyl iodide from plants was affected by the iodide concentration in the cultures. The recombinant RsHTMT demonstrated enzymatic properties similar to those of Brassica oleracea HTMT, especially in terms of its high specificity for iodide, bisulfide, and thiocyanate ions. A survey of biogenic emissions of methyl halides strongly suggests that the HTM/HTMT reaction is the key to understanding the biogenesis of methyl halides and methylated sulfur compounds in nature.
Assuntos
Metiltransferases/metabolismo , Raphanus/genética , Clonagem Molecular , Cromatografia Gasosa-Espectrometria de Massas , Genes de Plantas , Hidrocarbonetos Iodados/metabolismo , Metiltransferases/genética , Raphanus/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The enzymatic production of (S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with (S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum beta-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of (S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of (S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds.
Assuntos
Álcool Desidrogenase , Oxirredutases do Álcool , Biotecnologia/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Hidroxibutiratos/metabolismo , Actinomycetales/enzimologia , Actinomycetales/genética , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Redutase , Aldo-Ceto Redutases , Biocatálise , Hidroxibutiratos/química , Penicillium/enzimologia , Penicillium/genética , Engenharia de Proteínas/métodosRESUMO
In prokaryotes, menaquinone is used for respiration. In Escherichia coli, menaquinone is biosynthesized from chorismate by seven enzymes. However, very recently, we identified an alternative pathway (the futalosine pathway), which operates in some bacteria, including Streptomyces coelicolor, Helicobacter pylori, Campylobacter jejuni, and Thermus thermophilus. We describe the steps of this pathway, which branches at chorismate in a manner similar to the known pathway, but then follows a different route. This new pathway includes futalosine, an unusual nucleoside derivative consisting of inosine and o-substituted benzoate moieties, as a biosynthetic intermediate. In this study, a recombinant futalosine hydrolase (TTHA0556) of T. thermophilus, which participates in the second step of the pathway and catalyzes the reaction releasing hypoxanthine from futalosine, was prepared and used in functional analyses. Recombinant TTHA0556 formed a homotetramer and reacted only with futalosine; other structurally related nucleotides and nucleosides were not accepted. Recombinant TTHA0556 required no cofactors, and the optimum pH and temperature were 4.5 and 80 degrees C. The Km value was calculated to be 154.0+/-5.3 microM and the kcat value was 1.02/s. Recombinant TTHA0556 was slightly inhibited by hypoxanthine, with a Ki value of 1.1 mM.
Assuntos
Hidrolases/metabolismo , Nucleosídeos/metabolismo , Thermus thermophilus/enzimologia , Vitamina K 2/metabolismo , Animais , Bovinos , Concentração de Íons de Hidrogênio , Hidrolases/antagonistas & inibidores , Hidrolases/química , Hipoxantina/farmacologia , Cinética , Metais/farmacologia , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Temperatura , Thermus thermophilus/metabolismoRESUMO
Menaquinone is biosynthesized from chorismate via a new pathway involving 1,4-dihydroxy-6-naphthoate in Streptomyces and presumably in pathogenic bacteria Helicobacter and Campylobacter.
Assuntos
Ácido Corísmico/metabolismo , Naftóis/metabolismo , Vitamina K 2/metabolismo , Vitaminas/biossíntese , Campylobacter/metabolismo , Carbono/química , Radioisótopos de Carbono/química , Ácido Corísmico/química , Glucose/química , Helicobacter/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Químicos , Naftóis/química , Streptomyces/metabolismo , Vitamina K 2/química , Vitaminas/químicaRESUMO
BACKGROUND: The use of knowledge-based potential function is a powerful method for protein structure evaluation. A variety of formulations that evaluate single or multiple structural features of proteins have been developed and studied. The performance of functions is often evaluated by discrimination ability using decoy structures of target proteins. A function that can evaluate coarse-grained structures is advantageous from many aspects, such as relatively easy generation and manipulation of model structures; however, the reduction of structural representation is often accompanied by degradation of the structure discrimination performance. RESULTS: We developed a knowledge-based pseudo-energy calculating function for protein structure discrimination. The function (Discriminating Function using Main-chain Atom Coordinates, DFMAC) consists of six pseudo-energy calculation components that deal with different structural features. Only the main-chain atom coordinates of N, C alpha, and C atoms for the respective amino acid residues are required as input data for structure evaluation. The 231 target structures in 12 different types of decoy sets were separated into 154 and 77 targets, and function training and the subsequent performance test were performed using the respective target sets. Fifty-nine (76.6%) native and 68 (88.3%) near-native (< 2.0 A C alpha RMSD) targets in the test set were successfully identified. The average C alpha RMSD of the test set resulted in 1.174 with the tuned parameters. The major part of the discrimination performance was supported by the orientation-dependent component. CONCLUSION: Despite the reduced representation of input structures, DFMAC showed considerable structure discrimination ability. The function can be applied to the identification of near-native structures in structure prediction experiments.