RESUMO
OBJECTIVES: This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). RESULTS: L-amino acid deaminase from Proteus mirabilis was expressed in Escherichia coli BL21 (DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145/L341) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. CONCLUSIONS: The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.
Assuntos
Aminoácidos , Proteus mirabilis , Aminoácidos/metabolismo , Escherichia coli/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Proteus mirabilis/genéticaRESUMO
Coelenterazine (CTZ) is known as luciferin (a substrate) for the luminescence reaction with luciferase (an enzyme) in marine organisms and is unstable in aqueous solutions. The dehydrogenated form of CTZ (dehydrocoelenterazine, dCTZ) is stable and thought to be a storage form of CTZ and a recycling intermediate from the condensation reaction of coelenteramine and 4-hydroxyphenylpyruvic acid to CTZ. In this study, the enzymatic conversion of dCTZ to CTZ was successfully achieved using NAD(P)H:FMN oxidoreductase from the bioluminescent bacterium Vibrio fischeri ATCC 7744 (FRase) in the presence of NADH (the FRase-NADH reaction). CTZ reduced from dCTZ in the FRase-NADH reaction was identified by HPLC and LC/ESI-TOF-MS analyses. Thus, dCTZ can be enzymatically converted to CTZ in vitro. Furthermore, the concentration of dCTZ could be determined by the luminescence activity using the CTZ-utilizing luciferases (Gaussia luciferase or Renilla luciferase) coupled with the FRase-NADH reaction.
Assuntos
Aliivibrio fischeri/enzimologia , Proteínas de Bactérias/metabolismo , Imidazóis/metabolismo , Luciferases/metabolismo , NADH NADPH Oxirredutases/metabolismo , Pirazinas/metabolismo , Renilla/enzimologia , Aliivibrio fischeri/genética , Animais , Proteínas de Bactérias/genética , Biocatálise , Biotransformação , Cromatografia Líquida de Alta Pressão , Mononucleotídeo de Flavina/metabolismo , Expressão Gênica , Cinética , Luciferases/genética , Luminescência , Medições Luminescentes , NADH NADPH Oxirredutases/genética , Ácidos Fenilpirúvicos/metabolismo , Renilla/genéticaRESUMO
The efficiency of whole-cell biotransformation is often affected by the genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for the synthesis of phenylpyruvic acid (PPA) from l-phenylalanine. First, l-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into Escherichia coli BL21 (DE3) chromosome and the copy numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). Fifty-nine copies of pmLAAD were obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of isopropyl-ß-d-thiogalactopyranoside, the resulting strain, E. coli BL8â³recAâ³lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37% increase in PPA production compared to E. coli BL8â³recA. Finally, the engineered E. coli BL8â³recAâ³lacI strain achieved 19.14 g/L PPA at 24 h in a 5-L bioreactor.
Assuntos
Escherichia coli , Fenilalanina , Escherichia coli/genética , Escherichia coli/metabolismo , Fenilalanina/genética , Ácidos Fenilpirúvicos/metabolismo , Plasmídeos , Engenharia Metabólica/métodosRESUMO
Benzylamine is a commodity chemical used in the synthesis of motion-sickness treatments and anticonvulsants, in dyeing textiles, and as a precursor to the high-energy propellant CL-20. Because chemical production generates toxic waste streams, biosynthetic alternatives have been explored, recently resulting in a functional nine-step pathway from central metabolism (phenylalanine) in E. coli. We report a novel four-step pathway for benzylamine production, which generates the product from cellular phenylpyruvate using enzymes from different sources: a mandelate synthase (Amycolatopsis orientalis), a mandelate oxidase (Streptomyces coelicolor), a benzoylformate decarboxylase (Pseudomonas putida), and an aminotransferase (Salicibacter pomeroyi). This pathway produces benzylamine at 24 mg/L in 15 h (4.5% yield) in cultures of unoptimized cells supplemented with phenylpyruvate. Because the yield is low, supplementation with pathway intermediates is used to troubleshoot the design. This identifies conversion inefficiencies in the mandelate synthase-mediated synthesis of (S)-mandelic acid, and subsequent genome mining identifies a new mandelate synthase (Streptomyces sp. 1114.5) with improved yield. Supplementation experiments also reveal native redirection of ambient phenylpyruvate away from the pathway to phenylalanine. Overall, this work illustrates how retrosynthetic design can dramatically reduce the number of enzymes in a pathway, potentially reducing its draw on cellular resources. However, it also shows that such benefits can be abrogated by inefficiencies of individual conversions. Addressing these barriers can provide an alternative approach to green production of benzylamine, eliminating upstream dependence on chlorination chemistry.
Assuntos
Benzilaminas/metabolismo , Escherichia coli/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Proteínas de Bactérias/genética , Benzilaminas/química , Carboxiliases/genética , Escherichia coli/química , Escherichia coli/genética , Engenharia Metabólica/métodos , Família Multigênica , Oxirredutases/genética , Ácidos Fenilpirúvicos/química , Transaminases/genéticaRESUMO
Prolonged exposure to cold temperatures often results in a relatively low flowering rate in litchi (Litchi chinensis Sonn.) trees with younger leaves. This study aimed to verify the impact of stem girdling on litchi flowering by identifying and characterizing the induced metabolic changes. After a 60 day exposure to cold treatment at 15 °C/10 °C (12 h/12 h), the flowering rate of the girdled trees was 100%, while that of the non-girdled trees was 20%, indicating that girdling improved litchi flowering at its turning stage. The metabolic profiles of litchi leaves with and without stem girdling during floral induction were compared and 505 metabolites potentially associated with litchi flowering were detected. Most metabolites were involved in the metabolism of starch and sucrose, fatty acid, and phenylpyruvic acid. The metabolic pathways concerned with the biosynthesis of epinephrine, sucrose, and d-maltose were induced in leaves after girdling treatment. The level of galactitol, phenylpyruvic acid, acetyl-CoA, linoleic acid, alpha-linolenic acid, and 13-HPOT biosynthesis remained stable in the leaves from girdled trees but changed drastically in the leaves from non-girdled trees. In addition, 379 metabolites concerning flowering rate were characterized. Metabolism pathways of starch and sucrose, galactose, and linoleic acid are of great significance to the flowering of litchi. Linoleic acid exhibited the most significant variations between girdled trees and non-girdled trees with fold changes of up to 13.62. These results contribute to understanding the biological mechanism of litchi floral induction and the metabolic changes after stem girdling.
Assuntos
Litchi/metabolismo , Metaboloma , Folhas de Planta/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Ácido Linoleico/metabolismo , Litchi/crescimento & desenvolvimento , Ácidos Fenilpirúvicos/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Amido/metabolismo , Sacarose/metabolismoRESUMO
The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an l-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant l-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of α-keto acids exemplified for the synthesis of phenylpyruvic acid starting from l-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20â¯mM of substrate were converted after 4â¯h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme.
Assuntos
Hebeloma/metabolismo , L-Aminoácido Oxidase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Aminoácidos/metabolismo , Biocatálise , Biotransformação , Estabilidade Enzimática , L-Aminoácido Oxidase/genética , Especificidade por SubstratoRESUMO
To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⺠and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.
Assuntos
L-Lactato Desidrogenase , Lacticaseibacillus casei , Ácidos Fenilpirúvicos , Pichia , L-Lactato Desidrogenase/genética , Lacticaseibacillus casei/enzimologia , Lacticaseibacillus casei/genética , Ácidos Fenilpirúvicos/metabolismo , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses a cytosolic phenylalanine biosynthetic pathway intermediate, phenylpyruvate, as an amino acceptor for auxin formation.
Assuntos
Ácidos Indolacéticos/química , Ácidos Indolacéticos/metabolismo , Fenilalanina/biossíntese , Vias Biossintéticas/genética , Citosol/metabolismo , Indóis , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Plantas/metabolismo , TriptofanoRESUMO
AIM: 3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve 3-PLA yields in batch and continuous fermentation systems using of free-whole-cells of the three L. crustorum strains. MATERIALS AND RESULTS: The fermentation conditions of free-whole-cells of the three L. crustorum strains for 3-PLA production were optimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P < 0·05) greater 3-PLA production ability than the other strains after 10th recycle. The strain possesses three l-lactate dehydrogenase and three d-lactate dehydrogenase catalysing 3-PLA production from phenylpyruvic acid (PPA). Under the optimal conditions, the strain produced 15·2 mmol l-1 3-PLA (76% PPA conversion rate) in a batch fermentation system and 6·5 mmol l-1 h-1 3-PLA (55% PPA conversion rate) in a continuous fermentation system using a 0·6 dilution rate. CONCLUSIONS: Free-whole-cells of L. crustorum NWAFU 1078 showed excellent reusability and higher 3-PLA yields under optimal biotransformation conditions in both batch and continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the possibility to use the free-whole-cells of L. crustorum NWAFU 1078 as a biocatalyst for effective production of 3-PLA.
Assuntos
Lactatos/metabolismo , Lactobacillus/metabolismo , Técnicas de Cultura Celular por Lotes , Biotransformação , Fermentação , Ácidos Fenilpirúvicos/metabolismoRESUMO
2-Hydroxy acid dehydrogenases (2-HADHs) have been implicated in the synthesis of 2-hydroxy acids from 2-oxo acids that are used in wide areas of industry. d-lactate dehydrogenases (d-LDHs), a subfamily of 2-HADH, have been utilized to this purpose, yet they exhibited relatively low catalytic activity to the 2-oxo acids with large functional groups at C3. In this report, four putative 2-HADHs from Oenococcus oeni, Weissella confusa, Weissella koreensis and Pediococcus claussenii were examined for activity on phenylpyruvate (PPA), a substrate to 3-phenyllactic acid (PLA) with a C3 phenyl group. The 2-HADH from P. claussenii was found to have the highest kcat/Km on PPA with 1,348.03 s-1 mM-1 among the four enzymes with higher substrate preference for PPA than pyruvate. Sequential, structural and mutational analysis of the enzyme revealed that it belonged to the d-LDH family, and phenylalanine at the position 51 was the key residue for the PPA binding to the active site via hydrophobic interaction, whereas in the 2-HADHs from O. oeni and W. confusa the hydrophilic tyrosine undermined the interaction. Because phenyllactate is a potential precursor for pharmaceutical compounds, antibiotics and biopolymers, the enzyme could increase the efficiency of bio-production of valuable chemicals. This study suggests a structural basis for the high substrate preference of the 2-HADH, and further engineering possibilities to synthesize versatile 2-hydroxy acids.
Assuntos
Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Lactato Desidrogenases/química , Lactobacillales/enzimologia , Ácidos Fenilpirúvicos/metabolismo , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Hidroxiácidos/metabolismo , Cinética , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Lactatos/metabolismo , Lactobacillales/classificação , Lactobacillales/genética , Lactobacillales/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The 2-carboxy-6-hydroxyoctahydroindole (Choi) moiety is a hallmark of aeruginosins, a class of cyanobacterial derived bioactive linear tetrapeptides that possess antithrombotic activity. The biosynthetic pathway of Choi has yet to be resolved. AerE is a cupin superfamily enzyme that was shown to be involved in the biosynthesis of Choi, but its exact role remains unclear. This study reports the functional characterization and structural analyses of AerE. Enzymatic observation reveals that AerE can dramatically accelerate 1,3-allylic isomerization of the non-aromatic decarboxylation product of prephenate, dihydro-4-hydroxyphenylpyruvate (H2HPP). This olefin isomerization reaction can occur non-enzymatically and is the second step of the biosynthetic pathway from prephenate to Choi. The results of comparative structural analysis and substrate analogue binding geometry analysis combined with the results of mutational studies suggest that AerE employs an induced fit strategy to bind and stabilize the substrate in a particular conformation that is possibly favorable for 1,3-allylic isomerization of H2HPP through coordinate bonds, hydrogen bonds, π-π conjugation interaction and hydrophobic interactions. All of these interactions are critical for the catalytic efficiency.
Assuntos
Proteínas de Bactérias/química , Indóis/química , Isomerases/química , Microcystis/química , Oligopeptídeos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Ácidos Cicloexanocarboxílicos/química , Ácidos Cicloexanocarboxílicos/metabolismo , Cicloexenos/química , Cicloexenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Indóis/metabolismo , Isomerases/genética , Isomerases/metabolismo , Cinética , Microcystis/enzimologia , Modelos Moleculares , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ácidos Fenilpirúvicos/química , Ácidos Fenilpirúvicos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.
Assuntos
Vias Biossintéticas , Corismato Mutase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Plantas/metabolismo , Aminoácidos Dicarboxílicos/metabolismo , Ácidos Cicloexanocarboxílicos/metabolismo , Cicloexenos/metabolismo , Citosol/metabolismo , Plantas/genética , Plastídeos/genética , Plastídeos/metabolismo , Prefenato Desidratase/genética , Prefenato Desidratase/metabolismo , Transaminases/metabolismo , Sítio de Iniciação de Transcrição , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
L-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate L-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic L-amino acids with L-glutamine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and L-lysine being the best substrates. Methyl esters of these L-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 and 39 U mg-1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from L-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C.
Assuntos
Enzimas Imobilizadas/metabolismo , Hebeloma/enzimologia , L-Aminoácido Oxidase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , L-Aminoácido Oxidase/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.
Assuntos
Alcaloides/biossíntese , Oxirredutases/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Tropanos/metabolismo , Alcaloides/química , Atropa belladonna/química , Atropa belladonna/metabolismo , Estrutura Molecular , Oxirredutases/química , Oxirredutases/isolamento & purificação , Ácidos Fenilpirúvicos/química , Ácidos Fenilpirúvicos/isolamento & purificação , Tropanos/químicaRESUMO
To improve the specific activity and catalytic efficiency of l-LcLDH1, an NADH-dependent allosteric l-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1Q88R, Lcldh1I229A and Lcldh1T235G, were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, l-LcLDH1Q88R or l-LcLDH1I229A, displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded l-phenyllactic acid (PLA) with an enantiomeric excess (eep) more than 99%. Their catalytic efficiencies (kcat/Km) without d-fructose-1,6-diphosphate (d-FDP) were 4.8- and 5.2-fold that of l-LcLDH1. Additionally, the kcat/Km values of l-LcLDH1Q88R and l-LcLDH1I229A with d-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without d-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in l-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of l-LcLDH1.
Assuntos
Proteínas de Bactérias , L-Lactato Desidrogenase , Lacticaseibacillus casei/enzimologia , Ácidos Fenilpirúvicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Ácidos Fenilpirúvicos/química , Engenharia de ProteínasRESUMO
Phenyllactic acid (PLA) is a novel antimicrobial compound. A novel NADH-dependent d-lactate dehydrogenase (d-LDH), named as LF-d-LDH0653, with high phenylpyruvate (PPA) reducing activity was isolated from Lactobacillus fermentum JN248. Its optimum pH and temperature were 8.0 and 50 °C, respectively. The Michaelis-Menten constant (Km ), turnover number (kcat ), and catalytic efficiency (kcat /Km ) for NADH were 1.20 mmol/L, 67.39 s-1 , and 56.16 (mmol/L)-1 s-1 , respectively. The (Km ), (kcat ), and (kcat /Km ) for phenylpyruvate were 1.68 mmol/L, 122.66 s-1 , and 73.01 (mmol/L)-1 s-1 , respectively. This enzyme can catalyze phenylpyruvate and the product presented excellent optical purity (enantioselectivity >99%). The results suggest that LF-d-LDH0653 is a promising biocatalyst for the efficient synthesis of optically pure d-PLA. PRACTICAL APPLICATION: A novel d-LDH with phenylpyruvate reducing activity has been isolated and identified. It could be used as a reference for improving the production of optically pure d-PLA. d-PLA has a potential for application as antimicrobial an agent in dairy industry and baking industry, pharmaceutical agent in medicine and cosmetics.
Assuntos
Lactato Desidrogenases/metabolismo , Limosilactobacillus fermentum/enzimologia , Ácidos Fenilpirúvicos/metabolismo , Antibacterianos , Anti-Infecciosos , Catálise , Concentração de Íons de Hidrogênio , Lactato Desidrogenases/biossíntese , NAD/farmacologia , TemperaturaRESUMO
The development of new herbicides is receiving considerable attention to control weed biotypes resistant to current herbicides. Consequently, new enzymes are always desired as targets for herbicide discovery. 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is an enzyme engaged in photosynthetic activity and catalyzes the transformation of 4-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid (HGA). HPPD inhibitors constitute a promising area of discovery and development of innovative herbicides with some advantages, including excellent crop selectivity, low application rates, and broad-spectrum weed control. HPPD inhibitors have been investigated for agrochemical interests, and some of them have already been commercialized as herbicides. In this review, we mainly focus on the chemical biology of HPPD, discovery of new potential inhibitors, and strategies for engineering transgenic crops resistant to current HPPD-inhibiting herbicides. The conclusion raises some relevant gaps for future research directions.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/antagonistas & inibidores , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Agroquímicos , Inibidores Enzimáticos , Herbicidas , 4-Hidroxifenilpiruvato Dioxigenase/química , Agroquímicos/química , Agroquímicos/farmacologia , Arabidopsis/enzimologia , Catálise , Resistência a Medicamentos , Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Ácido Homogentísico/metabolismo , Estrutura Molecular , Ácidos Fenilpirúvicos/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Proteínas Recombinantes , Relação Estrutura-Atividade , Controle de Plantas DaninhasRESUMO
Streptomyces virginiae phenylpyruvate decarboxylase (PPDC) has not been identified before. Two putative branched-chain α-keto acid dehydrogenase subunit genes bkdC and bkdD from S. virginiae are similar to halves of other PPDC coding sequences. We cloned and characterized them biochemically in this work. The two proteins formed a stable complex attested by pull-down assay, consistent with the finding that their soluble expression was obtained only when they were coexpressed in Escherichia coli. The subunits were redesignated as SvPPDCα and SvPPDCß, because the SvPPDCα/ß complex catalyzed the conversion of phenylpyruvate to phenylacetaldehyde, reflecting the nature of the enzyme. Moreover, mutations of conserved residues in either of the two subunits led to inactivation or decreased specific activity of the enzymatic reaction. All previously identified PPDCs are encoded by a single gene. Here, we identified a new type of PPDC that contains two subunits, which gives new insights into the PPDC family.
Assuntos
Carboxiliases/genética , Carboxiliases/metabolismo , Domínio Catalítico/genética , Streptomycetaceae/enzimologia , Streptomycetaceae/genética , Sequência de Aminoácidos , Carboxiliases/química , Ativação Enzimática/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutação , Ácidos Fenilpirúvicos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de SequênciaRESUMO
Rosmarinic acid (RA) and its derivants are medicinal compounds that comprise the active components of several therapeutics. We isolated and characterised a tyrosine aminotransferase of Prunella vulgaris (PvTAT). Deduced PvTAT was markedly homologous to other known/putative plant TATs. Cytoplasmic localisation of PvTAT was observed in tobacco protoplasts. Recombinantly expressed and purified PvTAT had substrates preference for L-tyrosine and phenylpyruvate, with apparent K m of 0.40 and 0.48 mM, and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate. In vivo activity was confirmed by functional restoration of the Escherichia coli tyrosine auxotrophic mutant DL39. Agrobacterium rhizogenes-mediated antisense/sense expression of PvTAT in hairy roots was used to evaluate the contribution of PvTAT to RA synthesis. PvTAT were reduced by 46-95% and RA were decreased by 36-91% with low catalytic activity in antisense transgenic hairy root lines; furthermore, PvTAT were increased 0.77-2.6-fold with increased 1.3-1.8-fold RA and strong catalytic activity in sense transgenic hairy root lines compared with wild-type counterparts. The comprehensive physiological and catalytic evidence fills in the gap in RA-producing plants which didn't provide evidence for TAT expression and catalytic activities in vitro and in vivo. That also highlights RA biosynthesis pathway in P. vulgaris and provides useful information to engineer natural products.
Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Cinamatos/metabolismo , Depsídeos/metabolismo , Prunella/enzimologia , Prunella/metabolismo , Tirosina Transaminase/metabolismo , Agrobacterium/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Inativação Gênica , Teste de Complementação Genética , Cinética , Ácidos Fenilpirúvicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transformação Genética , Tirosina/metabolismo , Tirosina Transaminase/química , Tirosina Transaminase/isolamento & purificação , Ácido RosmarínicoRESUMO
Acceptor substrate specificity of Streptomyces roseochromogenes prenyltransferase SrCloQ was investigated using different non-genuine phenolic compounds. RP-UHPLC-UV-MSn was used for the tentative annotation and quantification of the prenylated products. Flavonoids, isoflavonoids and stilbenoids with different types of substitution were prenylated by SrCloQ, although with less efficiency than the genuine substrate 4-hydroxyphenylpyruvate. The isoflavan equol, followed by the flavone 7,4'-dihydroxyflavone, were the best non-genuine acceptor substrates. B-ring C-prenylation was in general preferred over A-ring C-prenylation (ratio 5:1). Docking studies of non-genuine acceptor substrates with the B-ring oriented towards the donor substrate dimethylallyl pyrophosphate, showed that the carbonyl group of the C-ring was able to make stabilizing interactions with the residue Arg160, which might determine the preference observed for B-ring prenylation. No reaction products were formed when the acceptor substrate had no phenolic hydroxyl groups. This preference can be explained by the essential hydrogen bond needed between a phenolic hydroxyl group and the residue Glu281. Acceptor substrates with an additional hydroxyl group at the C3' position (B-ring), were mainly O3'-prenylated (> 80% of the reaction products). This can be explained by the proximity of the C3' hydroxyl group to the donor substrate at the catalytic site. Flavones were preferred over isoflavones by SrCloQ. Docking studies suggested that the orientation of the B-ring and of the phenolic hydroxyl group at position C7 (A-ring) of flavones towards the residue Tyr233 plays an important role in this observed preference. Finally, the insights obtained on acceptor substrate specificity and regioselectivity for SrCloQ were extended to other prenyltransferases from the CloQ/NhpB family.