Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 148
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biosci Biotechnol Biochem ; 83(3): 456-462, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30445889

RESUMO

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.


Assuntos
Candida/enzimologia , Cetonas/química , Cetonas/metabolismo , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Pichia/enzimologia , Sequência de Aminoácidos , Biocatálise , Modelos Moleculares , Oxirredução , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
2.
J Adv Res ; 11: 15-22, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-30034872

RESUMO

The filamentous fungus Mortierella alpina 1S-4 is capable of accumulating a large amount of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs). Indeed, triacylglycerol production by M. alpina 1S-4 can reach 20 g/L of culture broth, and the critical cellular signaling and structural PUFA arachidonic acid (ARA) comprises 30%-70% of the total fatty acid. The demonstrated health benefits of functional PUFAs have in turn encouraged the search for rich sources of these compounds, including fungal strains showing enhanced production of specific PUFAs. Screening for mutants and targeted gene manipulation of M. alpina 1S-4 have elucidated the functions of various enzymes involved in PUFA biosynthesis and established lines with improved PUFA productivity. In some cases, these strains have been used for indistrial-scale production of PUFAs, including ARA. In this review, we described practical ARA production through mutant breeding, functional analyses of genes encoding enzymes involved in PUFA biosynthesis, and recent advances in the production of specific PUFAs through molecular breeding of M. alpina 1S-4.

3.
Bioorg Med Chem ; 26(7): 1327-1332, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28698052

RESUMO

A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.


Assuntos
Oxirredutases do Álcool/metabolismo , Ascomicetos/enzimologia , Fusarium/enzimologia , Isoleucina/análogos & derivados , Sequência de Aminoácidos , Biocatálise , Isoleucina/biossíntese , Isoleucina/química , Alinhamento de Sequência
4.
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
5.
J Gen Appl Microbiol ; 62(4): 174-80, 2016 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-27250664

RESUMO

One of the nitrile-synthesizing enzymes, ß-cyano-L-alanine synthase, catalyzes ß-cyano-L-alanine (ß-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the ß-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of ß-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine. This strategy can be used for the synthesis of labeled amino acids by using a carbon-labeled KCN as a substrate, resulting in an application for positron emission tomography.


Assuntos
Clonagem Molecular , Escherichia coli/genética , Liases/genética , Liases/metabolismo , Nitrilas/metabolismo , Pseudomonas/enzimologia , Rhodococcus/genética , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Aminoácidos/química , Aminoidrolases/genética , Asparagina/biossíntese , Ácido Aspártico/biossíntese , Escherichia coli/metabolismo , Expressão Gênica , Hidroliases/genética , Hidroliases/metabolismo , Tomografia por Emissão de Pósitrons , Cianeto de Potássio/metabolismo , Pseudomonas/genética , Rhodococcus/metabolismo , Especificidade por Substrato
6.
Appl Microbiol Biotechnol ; 100(13): 5747-57, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27188776

RESUMO

Biocatalysts (enzymes) have many advantages as catalysts for the production of useful compounds as compared to chemical catalysts. The stereoselectivity of the enzymes is one advantage, and thus the stereoselective production of chiral compounds using enzymes is a promising approach. Importantly, industrial application of the enzymes for chiral compound production requires the discovery of a novel useful enzyme or enzyme function; furthermore, improving the enzyme properties through protein engineering and directed evolution approaches is significant. In this review, the significance of several enzymes showing stereoselectivity (quinuclidinone reductase, aminoalcohol dehydrogenase, old yellow enzyme, and threonine aldolase) in chiral compound production is described, and the improvement of these enzymes using protein engineering and directed evolution approaches for further usability is discussed. Currently, enzymes are widely used as catalysts for the production of chiral compounds; however, for further use of enzymes in chiral compound production, improvement of enzymes should be more essential, as well as discovery of novel enzymes and enzyme functions.


Assuntos
Enzimas/química , Engenharia de Proteínas , Biocatálise , Evolução Molecular Direcionada , Enzimas/genética , Enzimas/metabolismo , Microbiologia Industrial , Especificidade por Substrato
7.
J Biosci Bioeng ; 122(1): 22-6, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26777235

RESUMO

We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase (Δ5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds. The isolation of nine Δ5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4.


Assuntos
Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Dessaturases/genética , Marcação de Genes , Engenharia Metabólica , Mortierella/genética , Mortierella/metabolismo , Ácido Araquidônico/análise , Ácido Araquidônico/biossíntese , Reatores Biológicos , Reparo do DNA por Junção de Extremidades , DNA Ligases/deficiência , DNA Ligases/genética , Ácidos Graxos Dessaturases/metabolismo , Mortierella/enzimologia
8.
J Biotechnol ; 208: 63-9, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26052021

RESUMO

The oil-producing zygomycete Mortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene that encodes for a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining. We disrupted the lig4 gene to improve the gene targeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4 strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the gene targeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute to basic research on the zygomycete.


Assuntos
DNA Ligases/genética , Proteínas Fúngicas/genética , Técnicas de Silenciamento de Genes , Marcação de Genes/métodos , Mortierella/genética , DNA Ligases/metabolismo , Proteínas Fúngicas/metabolismo , Mortierella/enzimologia
9.
J Biosci Bioeng ; 120(3): 299-304, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25845716

RESUMO

We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina, which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources.


Assuntos
Ácido Araquidônico/biossíntese , Embaralhamento de DNA , Ácidos Graxos Ômega-3/biossíntese , Mortierella/genética , Mortierella/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Mortierella/enzimologia , Temperatura , Transformação Genética
10.
Curr Genet ; 61(4): 579-89, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25782448

RESUMO

To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.


Assuntos
Ácido 8,11,14-Eicosatrienoico/metabolismo , DNA Fúngico/genética , DNA/genética , Marcação de Genes , Mortierella/genética , Ácido Araquidônico/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Fúngico/metabolismo , Ácidos Graxos Dessaturases/deficiência , Ácidos Graxos Dessaturases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Engenharia Genética , Recombinação Homóloga , Mesilatos/farmacologia , Mortierella/classificação , Mortierella/efeitos dos fármacos , Mortierella/metabolismo , Filogenia
11.
Chembiochem ; 16(3): 440-5, 2015 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-25639703

RESUMO

(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.


Assuntos
Cicloexanóis/síntese química , Cicloexanonas/síntese química , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Benzaldeídos/química , Benzaldeídos/metabolismo , Biocatálise , Candida/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Cicloexanonas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ligação de Hidrogênio , Dados de Sequência Molecular , Mutação , NADPH Desidrogenase/genética , Oxirredução , Conformação Proteica , Estereoisomerismo , Especificidade por Substrato
12.
J Biosci Bioeng ; 119(4): 410-5, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25283808

RESUMO

We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (αßγ structure), in which the α, ß, and γ subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (α,α'-dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50°C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30°C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate.


Assuntos
Acetaldeído/análogos & derivados , Aldeído Oxidase/metabolismo , Biocatálise , Burkholderia/enzimologia , Glicolatos/metabolismo , Acetaldeído/metabolismo , Aldeído Oxidase/antagonistas & inibidores , Aldeído Oxidase/química , Aldeídos/metabolismo , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Metais/farmacologia , Peso Molecular , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Temperatura , Xantina Desidrogenase/química
13.
J Lipid Res ; 55(9): 1855-63, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25002034

RESUMO

The PUFAs include many bioactive lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C20 PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C20 PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.


Assuntos
Ácido Araquidônico/metabolismo , Clostridium bifermentans/metabolismo , Ácido Eicosapentaenoico/metabolismo , Anaerobiose , Hidrogenação , Ácidos Linoleicos/metabolismo
14.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 6): 1695-703, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24914980

RESUMO

L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Šresolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Šoutwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.


Assuntos
Aeromonas/enzimologia , Glicina Hidroximetiltransferase/metabolismo , Mutação , Sequência de Bases , Domínio Catalítico , Primers do DNA , Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/genética , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , Especificidade por Substrato
15.
Biochem Biophys Res Commun ; 446(4): 911-5, 2014 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-24642255

RESUMO

(R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.


Assuntos
Agrobacterium tumefaciens/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Quinuclidinas/metabolismo , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , NAD/metabolismo , Conformação Proteica , Estereoisomerismo , Especificidade por Substrato
16.
AMB Express ; 4(1): 6, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24507746

RESUMO

Chiral molecule (R)-3-quinuclidinol, a valuable compound for the production of various pharmaceuticals, is efficiently synthesized from 3-quinuclidinone by using NADPH-dependent 3-quinuclidinone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of RrQR and the structure-based mutational analysis. The enzyme forms a tetramer, in which the core of each protomer exhibits the α/ß Rossmann fold and contains one molecule of NADPH, whereas the characteristic substructures of a small lobe and a variable loop are localized around the substrate-binding site. Modeling and mutation analyses of the catalytic site indicated that the hydrophobicity of two residues, I167 and F212, determines the substrate-binding orientation as well as the substrate-binding affinity. Our results revealed that the characteristic substrate-binding pocket composed of hydrophobic amino acid residues ensures substrate docking for the stereospecific reaction of RrQR in spite of its loose interaction with the substrate.

17.
J Biosci Bioeng ; 117(3): 275-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24099955

RESUMO

Some Prototheca spp. were previously reported to convert n-hexadecane to 5-hexadecanol and then to 5-hexadecanone through a unique subterminal oxidation pathway. Further analysis of derivatives derived from n-hexadecane indicated that Prototheca zopfii oxidized n-alkanes with C11 to C17 chain lengths at not only the 5th but also the 4th, 3rd and 2nd positions.


Assuntos
Alcanos/química , Carbono/química , Prototheca/metabolismo , Carbono/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Oxirredução , Prototheca/crescimento & desenvolvimento
18.
Appl Microbiol Biotechnol ; 98(1): 243-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23828603

RESUMO

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.


Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Candida/enzimologia , NADP/química , NADP/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Modelos Moleculares , Ligação Proteica , Conformação Proteica
19.
Proc Natl Acad Sci U S A ; 110(44): 17808-13, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24127592

RESUMO

In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Trato Gastrointestinal/microbiologia , Lactobacillus plantarum/enzimologia , Metabolismo dos Lipídeos/fisiologia , Redes e Vias Metabólicas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida , Clonagem Molecular , Primers do DNA/genética , Trato Gastrointestinal/metabolismo , Lactobacillus plantarum/metabolismo , Redes e Vias Metabólicas/genética , Camundongos , Dados de Sequência Molecular , Família Multigênica/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Análise de Sequência de DNA , Homologia de Sequência , Organismos Livres de Patógenos Específicos , Espectrometria de Massas em Tandem
20.
Biosci Biotechnol Biochem ; 77(8): 1650-4, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23924711

RESUMO

A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.


Assuntos
Fermentação/genética , Oxirredução , Pseudomonas putida/enzimologia , Ácido Pirúvico/química , Reatores Biológicos , Cruzamento , Imidas/química , Imidas/metabolismo , Cinética , Ácido Pirúvico/isolamento & purificação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...