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1.
Enzyme Microb Technol ; 141: 109667, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33051017

RESUMO

A number of organic synthesis involve threonine aldolase (TA), a pyridoxal phosphate (PLP)-dependent enzyme. Although the addition of exogenous PLP is necessary for the reactions, it increases the cost and complicates the purification of the product. This work constructed a PLP self-sufficient biocatalysis system for TA, which included an improvement of the intracellular PLP level and co-immobilization of TA with PLP. Engineered strain BL-ST was constructed by introducing PLP synthase PdxS/T to Escherichia coli BL21(ED3). The intracellular PLP concentration of the strain increased approximately fivefold to 48.5 µmol/gDCW. l-TA, from Bacillus nealsonii (BnLTA), was co-expressed in the strain BL-ST with PdxS/T, resulting in the engineered strain BL-BnLTA-ST. Compared with the control strain BL-BnLTA (254.1 U/L), the enzyme activity of the strain BL-BnLTA-ST reached 1518.4 U/L without the addition of exogenous PLP. An efficient co-immobilization system was then designed. The epoxy resin LX-1000HFA wrapped by polyethyleneimine (PEI) acted as a carrier to immobilize the crude enzyme solution of the strain BL-BnLTA-ST mixed with an extra 100 µM of exogenous PLP, resulting in the catalyst HFAPEI-BnLTA-STPLP 100. HFAPEI-BnLTA-STPLP 100 exhibited a half-life of approximately 450 h, and the application of the catalyst in the continuous biosynthesis of 3-[4-(methylsulfonyl) phenyl] serine had more than 180 batch reactions (>60%conv) without the extra addition of exogenous PLP. The excellent compatibility and stability of the system were further confirmed by other TAs. This work introduced a PLP self-sufficient biocatalysis system that can reduce the cost of PLP and contribute to the industrial application of TA. In addition, the system may also be applied in other PLP-dependent enzymes.


Assuntos
Enzimas Imobilizadas/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Fosfato de Piridoxal/metabolismo , Bacillus/enzimologia , Bacillus/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biocatálise , Meios de Cultura/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Resinas Epóxi/química , Escherichia coli/genética , Escherichia coli/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Glicina Hidroximetiltransferase/química , Glicina Hidroximetiltransferase/genética , Meia-Vida , Polietilenoimina/química , Fosfato de Piridoxal/biossíntese , Fosfato de Piridoxal/química
2.
Int J Mol Sci ; 21(17)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32825141

RESUMO

Plasmodium species are protozoan parasites causing the deadly malaria disease. They have developed effective resistance mechanisms against most antimalarial medication, causing an urgent need to identify new antimalarial drug targets. Ideally, new drugs would be generated to specifically target the parasite with minimal or no toxicity to humans, requiring these drug targets to be distinctly different from the host's metabolic processes or even absent in the host. In this context, the essential presence of vitamin B6 biosynthesis enzymes in Plasmodium, the pyridoxal phosphate (PLP) biosynthesis enzyme complex, and its absence in humans is recognized as a potential drug target. To characterize the PLP enzyme complex in terms of initial drug discovery investigations, we performed structural analysis of the Plasmodium vivax PLP synthase domain (Pdx1), glutaminase domain (Pdx2), and Pdx1-Pdx2 (Pdx) complex (PLP synthase complex) by utilizing complementary bioanalytical techniques, such as dynamic light scattering (DLS), X-ray solution scattering (SAXS), and electron microscopy (EM). Our investigations revealed a dodecameric Pdx1 and a monodispersed Pdx complex. Pdx2 was identified in monomeric and in different oligomeric states in solution. Interestingly, mixing oligomeric and polydisperse Pdx2 with dodecameric monodisperse Pdx1 resulted in a monodispersed Pdx complex. SAXS measurements revealed the low-resolution dodecameric structure of Pdx1, different oligomeric structures for Pdx2, and a ring-shaped dodecameric Pdx1 decorated with Pdx2, forming a heteromeric 24-meric Pdx complex.


Assuntos
Glutaminase/química , Simulação de Dinâmica Molecular , Plasmodium vivax/enzimologia , Multimerização Proteica , Proteínas de Protozoários/química , Sítios de Ligação , Glutaminase/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo , Fosfato de Piridoxal/biossíntese , Vitamina B 6/biossíntese
3.
J Biotechnol ; 321: 68-77, 2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-32445779

RESUMO

Pyridoxal 5'-phosphate (PLP) is an essential cofactor that participates in ∼4% enzymatic activities cataloged by the Enzyme Commission. The intracellular level of PLP is usually lower than that demanded in industrial catalysis. To realize the self-supply of PLP cofactor in whole-cell biotransformation, the de novo ribose 5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The pdxST genes from Bacillus subtilis 168 were introduced into the tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter or a compatible double-plasmid system. The 3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase with the increase in the intracellular PLP concentration in these strains with TPL and PdxST co-expression. Therefore, it is necessary to optimize the intracellular PLP metabolism level so as to achieve a higher L-DOPA titer and avoid the formation of L-DOPA-PLP cyclic adducts. The thi riboswitch binds to PLP and forms a complex such that the ribosome cannot have access to the Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST mRNA. Riboswitch was introduced into pET-TPL-pdxST-2 to downregulate the expression of PdxST and biosynthesis of PLP at the translation level by sequestering the ribosome-binding site. As a result, the titer and productivity of L-DOPA using the strain BL21-TPLST-Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively, with a catechol conversion of 95.9% and intracellular PLP accumulation of 24.8 µM.


Assuntos
Escherichia coli/genética , Levodopa , Fosfato de Piridoxal , Riboswitch/genética , Biotransformação , Escherichia coli/metabolismo , Levodopa/análise , Levodopa/genética , Levodopa/metabolismo , Fosfato de Piridoxal/biossíntese , Fosfato de Piridoxal/química , Fosfato de Piridoxal/genética , Fosfato de Piridoxal/metabolismo , Tirosina Fenol-Liase/química , Tirosina Fenol-Liase/genética , Tirosina Fenol-Liase/metabolismo
4.
J Cell Physiol ; 235(1): 504-512, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31506944

RESUMO

Pyridoxine/pyridoxamine 5'-phosphate oxidase (PNPO) and pyridoxal kinase (PDXK) cooperate to produce pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PDXK phosphorylates pyridoxine, pyridoxamine, and pyridoxal by producing PNP, PMP, and PLP, whereas PNPO oxidizes PNP, PMP, into PLP. We previously demonstrated that PDXK depletion in Drosophila and human cells impacts on glucose metabolism and DNA integrity. Here we characterized sgll, the Drosophila ortholog of PNPO gene, showing that its silencing by RNA interference elicits chromosome aberrations (CABs) in brains and induces diabetic hallmarks such as hyperglycemia and small body size. We showed that in sgllRNAi neuroblasts CABs are largely produced by the genotoxic effect of the advanced glycation end products triggered by high glucose. As in sgllRNAi cells, part of PLP is still produced by PDXK activity, these data suggest that PLP dosage need to be tightly regulated to guarantee glucose homeostasis and DNA integrity.


Assuntos
Drosophila melanogaster/metabolismo , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/biossíntese , Piridoxaminafosfato Oxidase/metabolismo , Animais , Aberrações Cromossômicas , DNA/fisiologia , Glucose/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Hiperglicemia/genética , Modelos Animais , Piridoxaminafosfato Oxidase/genética , Interferência de RNA , RNA Interferente Pequeno/genética
5.
Proc Natl Acad Sci U S A ; 116(48): 24164-24173, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31712440

RESUMO

PdxB (erythronate 4-phosphate dehydrogenase) is expected to be required for synthesis of the essential cofactor pyridoxal 5'-phosphate (PLP) in Escherichia coli Surprisingly, incubation of the ∆pdxB strain in medium containing glucose as a sole carbon source for 10 d resulted in visible turbidity, suggesting that PLP is being produced by some alternative pathway. Continued evolution of parallel lineages for 110 to 150 generations produced several strains that grow robustly in glucose. We identified a 4-step bypass pathway patched together from promiscuous enzymes that restores PLP synthesis in strain JK1. None of the mutations in JK1 occurs in a gene encoding an enzyme in the new pathway. Two mutations indirectly enhance the ability of SerA (3-phosphoglycerate dehydrogenase) to perform a new function in the bypass pathway. Another disrupts a gene encoding a PLP phosphatase, thus preserving PLP levels. These results demonstrate that a functional pathway can be patched together from promiscuous enzymes in the proteome, even without mutations in the genes encoding those enzymes.


Assuntos
Desidrogenases de Carboidrato/genética , Proteínas de Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Genoma Bacteriano , Fosfato de Piridoxal/biossíntese , Desidrogenases de Carboidrato/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Evolução Molecular Direcionada/métodos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Glucose/metabolismo , Redes e Vias Metabólicas/genética , Microrganismos Geneticamente Modificados , Mutação , Fosfato de Piridoxal/genética
6.
Sci Rep ; 9(1): 14188, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578392

RESUMO

In eukaryotes, pyridoxal kinase (PDXK) acts in vitamin B6 salvage pathway to produce pyridoxal 5'-phosphate (PLP), the active form of the vitamin, which is implicated in numerous crucial metabolic reactions. In Drosophila, mutations in the dPdxk gene cause chromosome aberrations (CABs) and increase glucose content in larval hemolymph. Both phenotypes are rescued by the expression of the wild type human PDXK counterpart. Here we expressed, in dPdxk1 mutant flies, four PDXK human variants: three (D87H, V128I and H246Q) listed in databases, and one (A243G) found in a genetic screening in patients with diabetes. Differently from human wild type PDXK, none of the variants was able to completely rescue CABs and glucose content elicited by dPdxk1 mutation. Biochemical analysis of D87H, V128I, H246Q and A243G proteins revealed reduced catalytic activity and/or reduced affinity for PLP precursors which justify this behavior. Although these variants are rare in population and carried in heterozygous condition, our findings suggest that in certain metabolic contexts and diseases in which PLP levels are reduced, the presence of these PDXK variants could threaten genome integrity and increase cancer risk.


Assuntos
Drosophila/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Piridoxal Quinase/genética , Fosfato de Piridoxal/genética , Animais , Animais Geneticamente Modificados/genética , Aberrações Cromossômicas , Drosophila/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Instabilidade Genômica , Glucose/metabolismo , Hemolinfa/metabolismo , Humanos , Larva/genética , Larva/metabolismo , Redes e Vias Metabólicas/genética , Mutação/genética , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/biossíntese , Vitamina B 6/biossíntese , Vitamina B 6/genética
7.
Environ Microbiol ; 20(1): 156-168, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29027347

RESUMO

Pyridoxal 5'-phosphate (PLP), the most important form of vitamin B6 serves as a cofactor for many proteins. Two alternative pathways for de novo PLP biosynthesis are known: the short deoxy-xylulose-5-phosphate (DXP)-independent pathway, which is present in the Gram-positive model bacterium Bacillus subtilis and the longer DXP-dependent pathway, which has been intensively studied in the Gram-negative model bacterium Escherichia coli. Previous studies revealed that bacteria contain many promiscuous enzymes causing a so-called 'underground metabolism', which can be important for the evolution of novel pathways. Here, we evaluated the potential of B. subtilis to use a truncated non-native DXP-dependent PLP pathway from E. coli for PLP synthesis. Adaptive laboratory evolution experiments revealed that two non-native enzymes catalysing the last steps of the DXP-dependent PLP pathway and two genomic alterations are sufficient to allow growth of vitamin B6 auxotrophic bacteria as rapid as the wild type. Thus, the existence of an underground metabolism in B. subtilis facilitates the generation of a pathway for synthesis of PLP using parts of a non-native vitamin B6 pathway. The introduction of non-native enzymes into a metabolic network and rewiring of native metabolism could be helpful to generate pathways that might be optimized for producing valuable substances.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Fosfato de Piridoxal/biossíntese , Fosfato de Piridoxal/metabolismo , Bacillus subtilis/enzimologia , Cisteína/análogos & derivados , Cisteína/metabolismo , Escherichia coli/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Pentosefosfatos/metabolismo , Proteínas , Vitamina B 6/metabolismo
8.
PLoS One ; 12(4): e0176374, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28448619

RESUMO

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for numerous enzymes involved in a diversity of cellular processes in living organisms. Previous analysis of the Actinobacillus pleuropneumoniae S-8 genome sequence revealed the presence of pdxS and pdxT genes, which are implicated in deoxyxylulose 5-phosphate (DXP)-independent pathway of PLP biosynthesis; however, little is known about their roles in A. pleuropneumoniae pathogenicity. Our data demonstrated that A. pleuropneumoniae could synthesize PLP by PdxS and PdxT enzymes. Disruption of the pdxS and pdxT genes rendered the pathogen auxotrophic for PLP, and the defective growth as a result of these mutants was chemically compensated by the addition of PLP, suggesting the importance of PLP production for A. pleuropneumoniae growth and viability. Additionally, the pdxS and pdxT deletion mutants displayed morphological defects as indicated by irregular and aberrant shapes in the absence of PLP. The reduced growth of the pdxS and pdxT deletion mutants under osmotic and oxidative stress conditions suggests that the PLP synthases PdxS/PdxT are associated with the stress tolerance of A. pleuropneumoniae. Furthermore, disruption of the PLP biosynthesis pathway led to reduced colonization and attenuated virulence of A. pleuropneumoniae in the BALB/c mouse model. The data presented in this study reveal the critical role of PLP synthases PdxS/PdxT in viability, stress tolerance, and virulence of A. pleuropneumoniae.


Assuntos
Actinobacillus pleuropneumoniae/enzimologia , Actinobacillus pleuropneumoniae/fisiologia , Ligases/metabolismo , Viabilidade Microbiana , Fosfato de Piridoxal/biossíntese , Estresse Fisiológico , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Feminino , Técnicas de Inativação de Genes , Peróxido de Hidrogênio/farmacologia , Ligases/deficiência , Ligases/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Cloreto de Sódio/farmacologia , Virulência
9.
Gene ; 587(1): 48-52, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-27106120

RESUMO

Vitamin B6 comprises six interconvertible pyridine compounds (vitamers), among which pyridoxal 5'-phosphate is a coenzyme involved in a high diversity of biochemical reactions. Humans and animals obtain B6 vitamers from diet, and synthesize pyridoxal 5'-phosphate by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. Currently, little is known on how pyridoxal 5'-phosphate biosynthesis is regulated, and pyridoxal 5'-phosphate is supplied to meet their requirement in terms of cofactor. Bombyx mori is a large silk-secreting insect, in which protein metabolism is most active, and the vitamin B6 demand is high. In this study, we successfully down-regulated the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase by body cavity injection of synthesized double-stranded small interfering RNA to 5th instar larvae of Bombyx mori, and analyzed the gene transcription levels of pyridoxal 5'-phosphate dependent enzymes, phosphoserine aminotransferase and glutamic-oxaloacetic transaminase. Results show that the gene expression of pyridoxal kinase and pyridoxine 5'-phosphate oxidase has a greater impact on the gene transcription of enzymes using pyridoxal 5'-phosphate as a cofactor in Bombyx mori. Our study suggests that pyridoxal 5'-phosphate biosynthesis and dynamic balance may be regulated by genetic networks.


Assuntos
Vias Biossintéticas , Bombyx/genética , Bombyx/metabolismo , Redes Reguladoras de Genes , Fosfato de Piridoxal/análogos & derivados , Vitaminas/biossíntese , Animais , Aspartato Aminotransferases/genética , Bombyx/enzimologia , Bombyx/crescimento & desenvolvimento , Larva/genética , Larva/metabolismo , Fosfato de Piridoxal/biossíntese , Piridoxaminafosfato Oxidase/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transaminases/genética , Transcrição Gênica , Transcriptoma
10.
Artigo em Inglês | MEDLINE | ID: mdl-26780217

RESUMO

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.


Assuntos
Bombyx/fisiologia , Hormônios de Inseto/fisiologia , Proteínas de Insetos/metabolismo , Piridoxal Quinase/metabolismo , Fosfato de Piridoxal/biossíntese , Piridoxaminafosfato Oxidase/metabolismo , Transcrição Gênica , Animais , Bombyx/efeitos dos fármacos , Bombyx/crescimento & desenvolvimento , China , Ecdisterona/antagonistas & inibidores , Ecdisterona/farmacologia , Ecdisterona/fisiologia , Corpo Adiposo/efeitos dos fármacos , Corpo Adiposo/crescimento & desenvolvimento , Corpo Adiposo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Hormônios de Inseto/antagonistas & inibidores , Hormônios de Inseto/farmacologia , Proteínas de Insetos/agonistas , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Hormônios Juvenis/farmacologia , Hormônios Juvenis/fisiologia , Cinética , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Piridoxal Quinase/antagonistas & inibidores , Piridoxal Quinase/química , Piridoxal Quinase/genética , Piridoxaminafosfato Oxidase/química , Piridoxaminafosfato Oxidase/genética , RNA Mensageiro/metabolismo , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/fisiologia , Sesquiterpenos/farmacologia , Transcrição Gênica/efeitos dos fármacos
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