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1.
Biochemistry ; 60(47): 3582-3595, 2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34747601

RESUMO

Temperature adaptation is ubiquitous among all living organisms, yet the molecular basis for this process remains poorly understood. It can be assumed that for parasite-host systems, the same enzymes found in both organisms respond to the same selection factor (human body temperature) with similar structural changes. Herein, we report the existence of a reversible temperature-dependent structural transition for the glycolytic enzyme lactate dehydrogenase (LDH) from the malaria parasite Plasmodium falciparum (pfLDH) and human heart (hhLDH) occurring in the temperature range of human fever. This transition is observed for LDHs from psychrophiles, mesophiles, and moderate thermophiles in their operating temperature range. Thermodynamic analysis reveals unique thermodynamic signatures of the LDH-substrate complexes defining a specific temperature range to which human LDH is adapted and parasite LDH is not, despite their common mesophilic nature. The results of spectroscopic analysis combined with the available crystallographic data reveal the existence of an active center within pfLDH that imparts psychrophilic structural properties to the enzyme. This center consists of two pockets, one formed by the five amino acids (5AA insert) within the substrate specificity loop and the other by the active site, that mutually regulate one another in response to temperature and induce structural and functional changes in the Michaelis complex. Our findings pave the way toward a new strategy for malaria treatments and drug design using therapeutic agents that inactivate malarial LDH selectively at a specific temperature range of the cyclic malaria paroxysm.


Assuntos
Aclimatação , Lactato Desidrogenases/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Temperatura Corporal , Temperatura Alta , Humanos , Lactato Desidrogenases/antagonistas & inibidores , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Miocárdio/enzimologia , Plasmodium falciparum/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Termodinâmica
2.
Bioengineered ; 12(1): 6459-6471, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34516347

RESUMO

D-lactate dehydrogenase (D-LDH) is widely used for the clinical detection of alanine aminotransferase (ALT) activity. It is a key enzyme in ALT detection kits, and its enzymatic properties directly determine sensitivity and accuracy of such kits. In this study, D-lactate dehydrogenase (WP_011543503, ldLDH) coding sequence derived from Lactobacillus delbrueckii was obtained from the NCBI database by gene mining. LdLDH was expressed and purified in Escherichia coli, and its enzyme activity, kinetic parameters, optimum temperature, and pH were characterized. Furthermore, stabilizers, including sugars, polyols, amino acids, certain salts, proteins, and polymers, were screened to improve stability of ldLDH during freeze-drying and storage. Finally, a kit based on ldLDH was tested to determine whether the enzyme had potential clinical applications. The results showed that ldLDH had a specific activity of 1,864 U/mg, Km value of 1.34 mM, optimal reaction temperature of 55°C, and an optimal pH between 7.0 and 7.5. When sucrose or asparagine was used as a stabilizer, freeze-dried ldLDH remained stable at 37°C for > 2 months without significant loss of enzymatic activity. These results indicated that ldLDH possesses high activity and stability. Test results using the ALT assay kit prepared with ldLDH were consistent with those of commercial kits, with a relative deviation <5%. These results indicated that ldLDH met the primary requirements for ALT assays, laying a foundation for the development of new ALT kits with potential clinical applications.


Assuntos
Alanina Transaminase , Análise Química do Sangue/métodos , Lactato Desidrogenases , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Humanos , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Lactobacillus delbrueckii/enzimologia , Lactobacillus delbrueckii/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Mol Biotechnol ; 63(10): 919-932, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34109551

RESUMO

Hydrophobins are small surface-active proteins. They can connect to hydrophobic or hydrophilic regions and oligomerize in solution to form massive construction. In nature, these proteins are produced by filamentous fungi at different stages of growth. So far, researchers have used them in various fields of biotechnology. In this study, recombinant hydrophobin-1 (rHFB1, 7.5 kDa) was used to stabilize recombinant D-lactate dehydrogenase (rD-LDH, 35 kDa). rD-LDH is a sensitive enzyme deactivated and oxidized by external agents such as O2 and lights. So, its stabilization with rHFB1 can be the best index to demonstrate the positive effect of rHFB1 on preserving and improving enzyme's activity. The unique ability of rHFB1 for interacting with hydrophobic regions of rD-LDH was predicted by protein-protein docking study with ClusPro and PIC servers and confirmed by fluorescence experiments, and Colorless Native-PAGE. Measurement of thermodynamic parameters allows for authenticating the role of rHFB1 as a thermal stabilizer in the protein-protein complex (rD-LDH@rHFB1). Interaction between rHFB1 and rD-LDH improved half-life of enzyme 2.25-fold at 40 °C. Investigation of the kinetic parameters proved that the presence of rHFB1 along with the rD-LDH enhancement strongly the affinity of the enzyme for pyruvate. Furthermore, an increase of Kcat/Km for complex displayed the effect of rHFB1 for improving the enzyme's catalytic efficiency.


Assuntos
Proteínas Fúngicas/metabolismo , Lactato Desidrogenases/química , Lactato Desidrogenases/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Interações Hidrofóbicas e Hidrofílicas , Lactato Desidrogenases/genética , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinâmica
4.
Clin Biochem ; 91: 45-51, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33617848

RESUMO

BACKGROUND: Lactate dehydrogenase (LDH) is a key enzyme that functions as a marker of cell damage. Its activity can be measured by a variety of laboratory methods. To eliminate inter-method bias and enhance equivalence among different measurement procedures, LDH detection needs to be standardized. METHODS: Optimized sequences coding for lactate dehydrogenase subunit A (LDH-A) and subunit B (LDH-B) were synthesized and cloned into the pRSFDuet vector, and then the constructed 6His-LDHA-pRSFDuet, 6His-LDHB-pRSFDuet, and 6His-LDHA-LDHB-pRSFDuet plasmids were transformed into Escherichia coli BL21 (DE3) for expression. The enzyme activity and specific activity of recombinant LDHs were detected. Electrophoresis of LDH isoenzymes was performed. The stability of recombinant LDHs and serum LDH was evaluated. Commutability of recombinant LDH-B was studied by the IFCC reference method and six routine methods. RESULTS: Three plasmids were constructed and three highly concentrated recombinant LDH isoenzymes were obtained. The specific activities of LDH-A, LDH-AB, and LDH-B were 18.08 U/mg, 21.74 U/mg, and 14.18 U/mg, respectively. There was a desirable linear correlation between the activities of recombinant LDH isoenzymes and their protein concentrations. Electrophoresis of LDH isoenzymes showed that the recombinant LDH-B corresponded to LDH1 and it demonstrated good stability at 4 °C and 25 °C for 5 weeks. LDH-B formulations in saline-bovine serum albumin solution and human serum matrix were commutable for six routine methods. CONCLUSION: Human recombinant LDH-B has great potential to become an improved and less expensive standard or reference material in external quality assessment for clinical LDH measurement.


Assuntos
Ensaios Enzimáticos Clínicos/normas , L-Lactato Desidrogenase , Lactato Desidrogenases , Humanos , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/normas , Lactato Desidrogenases/química , Lactato Desidrogenases/normas , Proteínas Recombinantes/química , Proteínas Recombinantes/normas , Padrões de Referência
5.
Protein Sci ; 29(12): 2387-2397, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33020946

RESUMO

During adaptive metabolic evolution a native glycerol dehydrogenase (GDH) acquired a d-lactate dehydrogenase (LDH) activity. Two active-site amino acid changes were detected in the altered protein. Biochemical studies along with comparative structure analysis using an X-ray crystallographic structure model of the protein with the two different amino acids allowed prediction of pyruvate binding into the active site. We propose that the F245S alteration increased the capacity of the glycerol binding site and facilitated hydrogen bonding between the S245 γ-O and the C1 carboxylate of pyruvate. To our knowledge, this is the first GDH to gain LDH activity due to an active site amino acid change, a desired result of in vivo enzyme evolution.


Assuntos
Bacillus , Proteínas de Bactérias , L-Iditol 2-Desidrogenase , Lactato Desidrogenases , Bacillus/enzimologia , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Cinética , L-Iditol 2-Desidrogenase/química , L-Iditol 2-Desidrogenase/genética , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Mutagênese Sítio-Dirigida
6.
Chembiochem ; 21(14): 1972-1975, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32175634

RESUMO

Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus-derived NAD-dependent d-lactate dehydrogenase (LhDLDH) to favor NCD by semirational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a "gate" guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant LhDLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency, and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD preference afforded much less d-lactate, thus suggesting the potential to construct NCD-mediated orthogonal metabolism.


Assuntos
Lactato Desidrogenases/metabolismo , NAD/biossíntese , Engenharia de Proteínas , Sequência de Aminoácidos , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Lactobacillus helveticus/enzimologia , Modelos Moleculares , Conformação Molecular , Mutação , NAD/química , Alinhamento de Sequência
7.
Enzyme Microb Technol ; 125: 37-44, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30885323

RESUMO

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 Substrato
8.
Arch Biochem Biophys ; 661: 178-186, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30472239

RESUMO

The most important mode of enzyme inactivation is thermal inactivation. Immobilization technology is an efficient approach to elongate the life-time of enzymes. d-lactate dehydrogenase (D-LDH) was stabilized at high temperatures with immobilization on CNT and fCNT. The kinetic and thermodynamic parameters, optimum temperature and pH, and the intrinsic fluorescence of free and immobilized enzymes were examined in the present study. Also, an attempt was made to investigate the effect of CNT and fCNT on the adsorption and conformation of d-lactate dehydrogenase using molecular dynamics (MD) simulations. In comparison with free enzyme, the immobilized enzyme displayed an improved stability at high temperatures and, therefore, the immobilized enzyme is suitable for use in the industry because most reactions in the industry happen at high temperatures. Results of the present study showed that the adsorption of enzyme on CNT is mediated through the van der Waals and π-π stacking interactions, whereas in the adsorption of enzyme on fCNT in addition to hydrophobic interactions, the hydrogen bonding between enzyme and functional groups of fCNT is involved. Moreover, RMSD, RMSF and secondary structure analysis indicate that the fCNT protects the conformation of enzyme more than CNT. Therefore, D-LDH can be efficiently immobilized upon the fCNT compared to the pristine CNT.


Assuntos
Enzimas Imobilizadas/química , Lactato Desidrogenases/química , Simulação de Dinâmica Molecular , Nanotubos de Carbono/química , Adsorção , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Temperatura Alta , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lactato Desidrogenases/metabolismo , Estrutura Secundária de Proteína , Solventes/química , Termodinâmica
9.
Int J Biol Macromol ; 119: 1171-1178, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30102986

RESUMO

d­Phenyllactic acid (d­PLA) shows great potential for biopreservative production owning to its anti-microbial activity. In this study, strain ZX1, which could inhibit the growth of other microbes was isolated and identified as Lactobacillus genus. Strain ZX1 could produce d­PLA with 0.16 g·L-1. Furthermore, a novel d­lactate dehydrogenase gene was identified and expressed in Escherichia coli BL21 (DE3) with the specific activity of 71.64 U·mg-1 protein under the optimal temperature and pH of 35 °C and 6.2. The kinetic constants for Kcat, Km, Vmax were 10.71 s-1, 2.356 mM and 11.27 µM·mg-1·min-1 for phenylpyruvic acid (PPA), respectively. 18.21 g·L-1d­PLA from PPA with yield of 90.49% and productivity of 2.49 g·L-1·h-1 was obtained by the recombinant E. coli BL21 harboring d­LDH, indicating that engineered E. coli BL21 (d­LDH) has excellent potential in commercial d­PLA production.


Assuntos
Engenharia Genética , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Lactatos/metabolismo , Lactobacillus/enzimologia , Sequência de Aminoácidos , Biotransformação , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Lactato Desidrogenases/química , Lactobacillus/genética , Metais/farmacologia , Temperatura
10.
Biochemistry ; 57(37): 5388-5406, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30149697

RESUMO

d-Lactate dehydrogenases (d-LDHs) from Fusobacterium nucleatum (FnLDH) and Escherichia coli (EcLDH) exhibit positive cooperativity in substrate binding, and the Pseudomonas aeruginosa enzyme (PaLDH) shows negatively cooperative substrate binding. The apo and ternary complex structures of FnLDH and PaLDH have been determined together with the apo-EcLDH structure. The three enzymes consistently form homotetrameric structures with three symmetric axes, the P-, Q-, and R-axes, unlike Lactobacillus d-LDHs, P-axis-related dimeric enzymes, although apo-FnLDH and EcLDH form asymmetric and distorted quaternary structures. The tetrameric structure allows apo-FnLDH and EcLDH to form wide intersubunit contact surfaces between the opened catalytic domains of the two Q-axis-related subunits in coordination with their asymmetric and distorted quaternary structures. These contact surfaces comprise intersubunit hydrogen bonds and hydrophobic interactions and likely prevent the domain closure motion during initial substrate binding. In contrast, apo-PaLDH possesses a highly symmetrical quaternary structure and partially closed catalytic domains that are favorable for initial substrate binding and forms virtually no intersubunit contact surface between the catalytic domains, which present their negatively charged surfaces to each other at the subunit interface. Complex FnLDH and PaLDH possess highly symmetrical quaternary structures with closed forms of the catalytic domains, which are separate from each other at the subunit interface. Structure-based mutations successfully converted the three enzymes to their dimeric forms, which exhibited no significant cooperativity in substrate binding. These observations indicate that the three enzymes undergo typical sequential allosteric transitions to exhibit their distinctive allosteric functions through the tetrameric structures.


Assuntos
Escherichia coli/enzimologia , Fusobacterium nucleatum/enzimologia , Lactato Desidrogenases/química , Pseudomonas aeruginosa/enzimologia , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Homologia de Sequência
11.
ACS Nano ; 12(8): 7911-7926, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30044604

RESUMO

Multistep enzymatic cascades are becoming more prevalent in industrial settings as engineers strive to synthesize complex products and pharmaceuticals in economical, environmentally friendly ways. Previous work has shown that immobilizing enzymes on nanoparticles can enhance their activity significantly due to localized interfacial effects, and this enhancement remains in place even when that enzyme's activity is coupled to another enzyme that is still freely diffusing. Here, we investigate the effects of displaying two enzymes with coupled catalytic activity directly on the same nanoparticle surface. For this, the well-characterized enzymes pyruvate kinase (PykA) and lactate dehydrogenase (LDH) were utilized as a model system; they jointly convert phosphoenolpyruvate to lactate in two sequential steps as part of downstream glycolysis. The enzymes were expressed with terminal polyhistidine tags to facilitate their conjugation to semiconductor quantum dots (QDs) which were used here as prototypical nanoparticles. Characterization of enzyme coassembly to two different sized QDs showed a propensity to cross-link into nanoclusters consisting of primarily dimers and some trimers. Individual and joint enzyme activity in this format was extensively investigated in direct comparison to control samples lacking the QD scaffolds. We found that QD association enhances LDH activity by >50-fold and its total turnover by at least 41-fold, and that this high activation appears to be largely due to stabilization of its quarternary structure. When both enzymes are simultaneously bound to the QD surfaces, their colocalization leads to >100-fold improvements in the overall rates of coupled activity. Experimental results in conjunction with detailed kinetic simulations provide evidence that this significant improvement in coupled activity is partially attributable to a combination of enhanced enzymatic activity and stabilization of LDH. More importantly, experiments aimed at disrupting channeled processes and further kinetic modeling suggest that the bulk of the performance enhancement arises from intermediary "channeling" between the QD-colocalized enzymes. A full understanding of the underlying processes that give rise to such enhancements from coupled enzymatic activity on nanoparticle scaffolds can provide design criteria for improved biocatalytic applications.


Assuntos
Lactato Desidrogenases/metabolismo , Nanopartículas/metabolismo , Piruvato Quinase/metabolismo , Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cinética , Lactato Desidrogenases/química , Lactobacillus leichmannii/enzimologia , Nanopartículas/química , Piruvato Quinase/química , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Propriedades de Superfície
12.
J Agric Food Chem ; 66(31): 8371-8381, 2018 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-30008205

RESUMO

d-Lactate dehydrogenase (d-LDH) catalyzes the reversible reaction pyruvate + NADH + H+ ↔ lactate + NAD+, which is a principal step in the production of d-lactate in lactic acid bacteria. In this study, we identified and characterized the major d-LDH (d-LDH1) from three d-LDHs in Leuconostoc mesenteroides, which has been extensively used in food processing. A molecular simulation study of d-LDH1 showed that the conformation changes during substrate binding. During catalysis, Tyr101 and Arg235 bind the substrates by hydrogen bonds and His296 acts as a general acid/base for proton transfer. These residues are also highly conserved and have coevolved. Point mutations proved that the substrate binding sites and catalytic site are crucial for enzyme activity. Network and phylogenetic analyses indicated that d-LDH1 and the homologues are widely distributed but are most abundant in bacteria and fungi. This study expands the understanding of the functions, catalytic mechanism, and evolution of d-LDH.


Assuntos
Lactato Desidrogenases/metabolismo , Ácido Láctico/biossíntese , Lactobacillales/enzimologia , Sítios de Ligação , Catálise , Domínio Catalítico , Sequência Conservada , Evolução Molecular , Manipulação de Alimentos/métodos , Ligação de Hidrogênio , Lactato Desidrogenases/química , Leuconostoc mesenteroides/enzimologia , Mutação Puntual , Ligação Proteica , Conformação Proteica
13.
J Biosci Bioeng ; 126(4): 425-430, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29691195

RESUMO

A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03-2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/química , Técnicas Biossensoriais/métodos , Lactato Desidrogenases/química , Ácido Láctico/química , Archaea/química , Archaea/genética , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Técnicas Biossensoriais/instrumentação , Eletrodos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Ácido Láctico/metabolismo
14.
Chemphyschem ; 18(13): 1811-1821, 2017 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-28467664

RESUMO

Signal-controlled release of DNA from Fe3+ -cross-linked alginate hydrogel electrochemically deposited on an electrode surface was studied. The multiple input signals were logically processed with the help of the enzyme-biocatalyzed reactions. Boolean logic gates, OR, AND, INH, were realized with the biocatalytic reactions performed by the enzymes entrapped in the alginate film. Hydrogen peroxide produced by the enzymatic reactions resulted in the degradation of the alginate hydrogel and DNA release. The alginate degradation was facilitated by the formation of free radicals in the Fenton-type reaction catalyzed by iron cations cross-linking the alginate hydrogel. The studied approach is versatile and can be adapted to various chemical signals processed by various enzymes with differently implemented Boolean logic. This work illustrates a novel concept of functional integration of biomolecular computing and actuation.


Assuntos
Alginatos/química , Computadores Moleculares , Reagentes de Ligações Cruzadas/química , DNA/metabolismo , Compostos Férricos/química , Lógica , Animais , Biocatálise , DNA/química , Esterases/química , Esterases/metabolismo , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Lactato Desidrogenases/química , Lactato Desidrogenases/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Dióxido de Silício/química , Dióxido de Silício/metabolismo
15.
Biochemistry ; 56(19): 2488-2496, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28445027

RESUMO

We present new findings about how primary and secondary structure affects the role of fast protein motions in the reaction coordinates of enzymatic reactions. Using transition path sampling and committor distribution analysis, we examined the difference in the role of these fast protein motions in the reaction coordinate of lactate dehydrogenases (LDHs) of Apicomplexa organisms Plasmodium falciparum and Cryptosporidium parvum. Having evolved separately from a common malate dehydrogenase ancestor, the two enzymes exhibit several important structural differences, notably a five-amino acid insertion in the active site loop of P. falciparum LDH. We find that these active site differences between the two organisms' LDHs likely cause a decrease in the contribution of the previously determined LDH rate-promoting vibration to the reaction coordinate of P. falciparum LDH compared to that of C. parvum LDH, specifically in the coupling of the rate-promoting vibration and the hydride transfer. This effect, while subtle, directly shows how changes in structure near the active site of LDH alter catalytically important motions. Insights provided by studying these alterations would prove to be useful in identifying LDH inhibitors that specifically target the isozymes of these parasitic organisms.


Assuntos
Cryptosporidium parvum/enzimologia , Lactato Desidrogenases/metabolismo , Modelos Moleculares , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Biocatálise , Domínio Catalítico , Biologia Computacional , Bases de Dados de Proteínas , Evolução Molecular , Ligação de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Simulação de Dinâmica Molecular , Mutagênese Insercional , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Teoria Quântica
16.
J Am Chem Soc ; 138(45): 15004-15010, 2016 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-27766841

RESUMO

Transition path sampling simulations have proposed that human heart lactate dehydrogenase (LDH) employs protein promoting vibrations (PPVs) on the femtosecond (fs) to picosecond (ps) time scale to promote crossing of the chemical barrier. This chemical barrier involves both hydride and proton transfers to pyruvate to form l-lactate, using reduced nicotinamide adenine dinucleotide (NADH) as the cofactor. Here we report experimental evidence from three types of isotope effect experiments that support coupling of the promoting vibrations to barrier crossing and the coincidence of hydride and proton transfer. We prepared the native (light) LDH and a heavy LDH labeled with 13C, 15N, and nonexchangeable 2H (D) to perturb the predicted PPVs. Heavy LDH has slowed chemistry in single turnover experiments, supporting a contribution of PPVs to transition state formation. Both the [4-2H]NADH (NADD) kinetic isotope effect and the D2O solvent isotope effect were increased in dual-label experiments combining both NADD and D2O, a pattern maintained with both light and heavy LDHs. These isotope effects support concerted hydride and proton transfer for both light and heavy LDHs. Although the transition state barrier-crossing probability is reduced in heavy LDH, the concerted mechanism of the hydride-proton transfer reaction is not altered. This study takes advantage of triple isotope effects to resolve the chemical mechanism of LDH and establish the coupling of fs-ps protein dynamics to barrier crossing.


Assuntos
Deutério/metabolismo , Coração , Hidrogênio/metabolismo , Lactato Desidrogenases/metabolismo , Prótons , Deutério/química , Humanos , Hidrogênio/química , Cinética , Lactato Desidrogenases/química , NAD/química , NAD/metabolismo , Vibração
17.
Chembiochem ; 17(16): 1491-4, 2016 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-27237045

RESUMO

High production of polymer-grade d-lactic acid is urgently required, particularly for the synthesis of polylactic acid. High-temperature fermentation has multiple advantages, such as lower equipment requirement and energy consumption, which are essential for lowering operating costs. We identified and introduced a unique d-lactate dehydrogenase into a thermotolerant butane-2,3-diol-producing strain. Carbon flux "trapping" was achieved by a "trapping point" created by combination of the introduced enzyme and the host efflux pump, which afforded irreversible transport of d-lactic acid. The overall carbon flux of the engineered strain was significantly enhanced and was redistributed predominantly to d-lactic acid. Under optimized conditions at 50 °C, d-lactic acid reached the highest titer (226.6 g L(-1) ) reported to date. This discovery allows us to extend the carbon flux trapping strategy to engineering complex metabolic networks.


Assuntos
Ciclo do Carbono , Lactato Desidrogenases/metabolismo , Ácido Láctico/biossíntese , Polímeros/metabolismo , Temperatura , Lactato Desidrogenases/química , Ácido Láctico/química , Polímeros/química , Fatores de Tempo
18.
Int J Biol Macromol ; 86: 155-61, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26802246

RESUMO

Ionic liquids (ILs) were widely used in scientific and industrial application and have been reported to possess potential toxicity to the environment and human health. The effects of six typical N-methylimidazolium-based ILs ([Cnmim]X, n=4, 6, 8; X=Br(-), Cl(-), BF4(-), CF3SO3(-)) on the lactic dehydrogenase (LDH) activity and the molecular interaction mechanism of ILs and the LDH were investigated with the aid of spectroscopic techniques. Experimental results showed that the LDH activity was inhibited in the presence of ILs. For the ILs with the same anion but different cations, their inhibitory ability on the LDH activity increased with increasing the alkyl chain length on the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of LDH with the addition of ILs. Both positive ΔH and ΔS suggested that hydrophobicity was the major driven force in the interaction process as expected.


Assuntos
Inibidores Enzimáticos/farmacologia , Líquidos Iônicos/farmacologia , Lactato Desidrogenases/antagonistas & inibidores , Interações Hidrofóbicas e Hidrofílicas , Lactato Desidrogenases/química , Lactato Desidrogenases/metabolismo , Conformação Proteica , Termodinâmica
19.
Wei Sheng Wu Xue Bao ; 56(11): 1811-8, 2016 Nov 04.
Artigo em Chinês | MEDLINE | ID: mdl-29741845

RESUMO

Objective: Sporolactobacillus inulinus, a typical homofermentative lactic acid bacterium, is an efficient D-lactic acid producer. Various environment factors affect the productivity of S. inulinus. Glucokinase, phosphofructokinase, pyruvate kinase and lactic dehydrogenase are the key enzymes of D-lactic acid production from glucose by S. inulinus. The characteristics of these enzymes are important in controlling and regulating the fermentation process. According to the genome bioinformatics analysis of S. inulinus CASD, three putative D-lactate dehydrogenases were identified, among which the bifunctional protein had been reported. In this study, we provided insights into the characteristics of the other two D-lactate dehydrogenase isozymes. Methods: S. inulinus Y2-8 genome was used as the template to amplify D-lactate dehydrogenase gene (dldh) and D-isomer specific 2-hydroxyacid dehydrogenase gene (dhdh). The two recombinant strains E-pET-28a/dldh and E-pET-28a/dhdh were constructed for enzyme expression. Both recombinants DLDH and DHDH could convert pyruvic acid into D-lactic acid. Results: Enzymes expressed by recombinant strains were purified by Ni-NTA chromatography. The apparent molecular mass of DLDH was approximately 37 kDa by SDS-PAGE analysis, and DLDH showed a high affinity to pyruvate with the Km value of (0.58±0.04) mmol/L. The optimal reaction temperature and pH for DLDH was 35℃ and 6.5, respectively. The apparent molecular mass of DHDH was approximately 39 kDa, and the Km of DHDH toward pyruvate was (1.70±0.08) mmol/L. The optimum catalysis temperature and pH of DHDH were 30℃ and 7.5, respectively. Conclusion: According to the Km and optimal reaction pH, DLDH was suggested as the main catalyst in formation D-lactic acid from pyruvate during the fermentation. The enzymatic properties would contribute to the regulation of the fermentation of S. inulinus.


Assuntos
Bacillales/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lactato Desidrogenases/química , Lactato Desidrogenases/metabolismo , Ácido Láctico/metabolismo , Bacillales/química , Bacillales/genética , Proteínas de Bactérias/genética , Estabilidade Enzimática , Glucose/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lactato Desidrogenases/genética , Ácido Pirúvico/metabolismo
20.
J Mol Endocrinol ; 55(3): 219-29, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26307561

RESUMO

Gestational diabetes mellitus (GDM) is a condition commonly encountered during mid to late pregnancy with pathologic manifestations including hyperglycemia, hyperinsulinemia, insulin resistance, and fetal mal-development. The deficit and dysfunction of insulin secreting ß-cells are signature symptoms for GDM. Pancreatic progenitors derived from human embryonic stem cells (hESCs) were shown to be able to effectively treat diabetes in mice. In this study, we first identified that microRNA-410 (miR-410) directly targets lactate dehydrogenase A (LDHA), a gene selectively repressed in normal insulin secreting ß-cells. hESCs that can be induced to express miR-410 hence keeping LDHA levels in check were then differentiated in vitro into pancreatic endoderm, followed by transplantation into db/+ mouse model of GDM. The transplant greatly improved glucose metabolism and reproductive outcome of the pregnant females suffering from GDM. Our findings describe for the first time the method of combining miRNA with hESCs, providing proof of concept by employing genetically modified stem cell therapy for treating GDM.


Assuntos
Diferenciação Celular , Diabetes Gestacional/genética , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , MicroRNAs/genética , Transplante de Pâncreas , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Biomarcadores , Composição Corporal , Linhagem Celular , Diabetes Gestacional/sangue , Diabetes Gestacional/terapia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/genética , Hiperinsulinismo/sangue , Hiperinsulinismo/genética , Hiperinsulinismo/terapia , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Camundongos , MicroRNAs/química , Gravidez , Interferência de RNA , Reprodução
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