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1.
J Agric Food Chem ; 69(42): 12485-12493, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34657425

RESUMO

L-Tryptophan is known as an aromatic amino acid and one of the essential amino acids that must be ingested through various additives or food. TrpCF is a bifunctional enzyme that has indole-glycerol-phosphate synthase (IGPS) and phosphoribosylanthranilate isomerase (PRAI) activity. In this report, we identified the crystal structure of TrpCF from Corynebacterium glutamicum (CgTrpCF) and successfully elucidated the active site by attaching rCdRP similar to the substrate and product of the TrpCF reaction. Also, we revealed that CgTrpCF shows a conformational change at the loops upon substrate binding. We analyzed amino acid sequences of the homologues of CgTrpCF, and the residues of the substrate-binding site in TrpCF were highly conserved except for some residues. These less conserved residues were replaced by site-directed mutagenesis experiments. Consequently, we obtained the CgTrpCFP294K (PRAICD/P294K) variant that has enhanced activity.


Assuntos
Aldose-Cetose Isomerases , Corynebacterium glutamicum , Aldose-Cetose Isomerases/genética , Corynebacterium glutamicum/genética , Indol-3-Glicerolfosfato Sintase , Isomerases
2.
J Invertebr Pathol ; 185: 107669, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34560108

RESUMO

The pathogenicity of a common phytopathogenic bacterium, Pseudomonas syringae, against animal model hosts, such as mice and Caenorhabditis elegans, has been recently revealed. However, most of the virulence determinants associated with pathogenesis remain elusive. In the current study, we performed predictive analysis of virulence factors against C. elegans in the genome of the wild-type P. syringae strain MB03. Nine predicted nematicidal proteins were expressed and purified in recombinant Escherichia coli strains and were evaluated to define their toxicity against C. elegans in liquid killing assays. Next, we focused on one essential 2-methyl citrate cycle protein, PrpF03, which showed the highest lethal activity against C. elegans compared to the other tested proteins with a half lethal concentration (LC50) of 155.3 (123.4-176.6) µg mL-1 and a half lethal time (LT50) of 3.72 (1.64-4.85) days. Purified PrpF03 also caused adverse effects on the brood size, growth, and motility of C. elegans. Moreover, the PrpF03 protein exhibited pathological activity towards the intestinal tract of C. elegans. We surmise that the PrpF03 protein functions as a virulence factor when it blocks the average circulation of the 2-methyl citrate cycle of C. elegans by accumulating 2-methyl citrate in the gut of C. elegans, which damages and restrains the growth of intestinal tissues that ultimately kill C. elegans. The discovery of specific nematicidal activities of PrpF03 provides a better understanding of the mechanisms of phytopathogenic P. syringae against nematodes and could aid in developing nematode pest-controlling agents in agriculture.


Assuntos
Antinematódeos/farmacologia , Proteínas de Bactérias/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Isomerases/farmacologia , Pseudomonas syringae/enzimologia , Fatores de Virulência/genética , Animais
3.
J Agric Food Chem ; 69(29): 8160-8171, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34281339

RESUMO

A linoleate isomerase complex including myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase, and acetoacetate decarboxylase has been confirmed as the pivotal factor for conjugated linoleic acid (CLA) production in Lactobacillus plantarum. However, its role in the metabolism and health-associated benefits of Lactobacillus remain unclear. In the current study, the mild type, knockout, and complemented mutants of the linoleate isomerase complex of L. plantarum ZS2058 were used to investigate those putative effects. The metabonomic results showed that a linoleate isomerase complex could significantly influence the glycol-metabolism, lipid metabolism, and antioxidant compounds. Especially, with the stress of linoleic acid, linoleate isomerase complex knockout mutants induced the increase of several antioxidant compounds, such as glutamic acid, glycine, l-cysteine, glycerol, and l-sorbosone. Moreover, the linoleate isomerase complex played a pivotal role in ameliorating DSS-induced colitis. The knockout mutants showed effects similar to those in the DSS group, whereas complementation of the corresponding gene in the knockout mutants could restore the anti-inflammatory activity, wherein the integrity of a mucus layer was repaired, the level of pro-inflammatory cytokines decreased, and the amount of anti-inflammatory cytokines increased significantly. All the results indicated that the linoleate isomerase complex plays a key role in CLA production and metabolism as well as the health-associated benefits of L. plantarum ZS2058. These results are conducive to promote clinical trials and product development of probiotics for colitis.


Assuntos
Colite , Lactobacillus plantarum , Probióticos , Animais , Sulfato de Dextrana , Isomerases/genética , Lactobacillus , Lactobacillus plantarum/genética , Ácido Linoleico , Camundongos
4.
J Chem Theory Comput ; 17(7): 4524-4537, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34087064

RESUMO

Selection of residues and other molecular fragments for inclusion in the quantum mechanics (QM) region for QM/molecular mechanics (MM) simulations is an important step for these calculations. Here, we present an approach that combines protein sequence/structure evolution and electron localization function (ELF) analyses. The combination of these two analyses allows the determination of whether a residue needs to be included in the QM subsystem or can be represented by the MM environment. We have applied this approach on two systems previously investigated by QM/MM simulations, 4-oxalocrotonate tautomerase (4OT) and ten-eleven translocation-2 (TET2), that provide examples where fragments may or may not need to be included in the QM subsystem. Subsequently, we present the use of this approach to determine the appropriate QM subsystem to calculate the minimum energy path (MEP) for the reaction catalyzed by human DNA polymerase λ (Polλ) with a third cation in the active site. Our results suggest that the combination of protein evolutionary and ELF analyses provides insights into residue/molecular fragment selection for QM/MM simulations.


Assuntos
Evolução Química , Isomerases/química , Teoria Quântica , Sequência de Aminoácidos , Simulação de Dinâmica Molecular , Pseudomonas putida/enzimologia
5.
Biochemistry ; 60(22): 1776-1786, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34019384

RESUMO

The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple ß-α-ß structural scaffold. Most members are constructed from a single-core ß-α-ß motif or two consecutively fused ß-α-ß motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.


Assuntos
Hidrolases/química , Isomerases/química , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Catálise , Domínio Catalítico/fisiologia , Evolução Molecular , Cinética , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos
6.
Org Lett ; 23(8): 3162-3166, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33826848

RESUMO

The sesquiterpene cyclases pentalenene synthase (PenA) and two Δ6-protoilludene synthases Omp6 and Omp7 convert a FPP ether into several new tetrahydrofurano terpenoids, one of which is also formed as the main product by the sesquiterpene cyclase BcBOT2. Thus, PenA, Omp6/7, and BcBOT2 follow closely related catalytic pathways and induce similar folding of their diphosphate substrates despite low levels of amino acid sequence similarity. Some of the new terpenoids show pronounced olfactoric properties.


Assuntos
Carbono-Carbono Liases/química , Éter/química , Isomerases/química , Sequência de Aminoácidos , Estrutura Molecular , Sesquiterpenos/química
7.
Int J Biol Macromol ; 182: 154-161, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33836196

RESUMO

Natural [4 + 2]-cyclases catalyze concerted cycloaddition during biosynthesis of over 400 natural products reported. Microbial [4 + 2]-cyclases are structurally diverse with a broad range of substrates. Thus far, about 52 putative microbial [4 + 2]-cyclases of 13 different types have been characterized, with over 20 crystal structures. However, how these cyclases have evolved during natural product biosynthesis remains elusive. Structural and phylogenetic analyses suggest that these different types of [4 + 2]-cyclases might have diverse evolutionary origins, such as reductases, dehydratases, methyltransferases, oxidases, etc. Divergent evolution of enzyme function might have occurred in these different families. Understanding the independent evolutionary history of these cyclases would provide new insights into their catalysis mechanisms and the biocatalyst design.


Assuntos
Proteínas de Bactérias/genética , Evolução Molecular , Compostos Heterocíclicos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência Conservada , Isomerases/química , Isomerases/genética , Isomerases/metabolismo , Ligases/química , Ligases/genética , Ligases/metabolismo , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Homologia de Sequência
8.
Planta ; 253(5): 107, 2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33866441

RESUMO

MAIN CONCLUSION: Importation of taxadiene synthase into chloroplasts is important for the efficient heterologous production of taxadiene. Taxadiene, the first committed precursor to taxol, is synthesized from geranylgeranyl pyrophosphate (GGPP) by action of taxadiene synthase (TS). Heterologous production of taxadiene could potentially rely on both cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. We suggest the compartmentalized engineering in chloroplast as an efficient approach for taxadiene production. In this study, we directly introduced the TS gene from Taxus brevifolia into the tobacco chloroplast genome and found that the transplastomic plants accumulated a low content of taxadiene, ~ 5.6 µg/g dry weight (DW). Moreover, we tried a combination of MEP and MVA pathways for taxadiene synthesis by nuclear transformation with a truncated version of TS (without encoding a transit peptide) into the transplastomic plants. However, this did not further improve the taxadiene production. In contrast, we found that taxadiene could be produced up to 87.8 µg/g DW in leaves of transgenic plants expressing TS with a chloroplast transit peptide, which was significantly higher than that in leaves of transplastomic plants. Thus, this study highlights the importance of TS importation into chloroplast for production of taxadiene.


Assuntos
Isomerases , Tabaco , Alcenos , Cloroplastos/genética , Diterpenos , Tabaco/genética
9.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920773

RESUMO

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.


Assuntos
Proteínas Fúngicas/metabolismo , Hypocreales/metabolismo , Hypocreales/patogenicidade , Isomerases/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteômica , Sequência de Aminoácidos , Proteínas Fúngicas/química , Deleção de Genes , Genoma Fúngico , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Isomerases/química , Micotoxinas/genética , Micotoxinas/metabolismo , Proteoma/metabolismo , Spliceossomos/metabolismo , Esporos Fúngicos/metabolismo , Estresse Fisiológico , Frações Subcelulares/metabolismo
10.
Eur J Med Chem ; 216: 113308, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33713976

RESUMO

Unique structure, characteristic reactivity, and facile synthesis of metal complexes have made them efficient ligands in drug development research. Among them, rhodium complexes have a limited history and there are a few discussions about their biological activities documented in the literature. However, investigation of kinetically inert rhodium complexes has recently attracted lots of attention and especially there are various evidences on their anti-cancer activity. It seems that they can be investigated as a versatile surrogates or candidates for the existing drugs which do not affect selectively or suffer from various side effects. In recent years, there has been an increasing interest in the use of mononuclear rhodium (III) organometallo drugs due to its versatile structurally important aspects to inhibit various enzymes. It has been demonstrated that organometallic Rh complexes profiting from both organic and inorganic aspects have shown more potent biological activities than classical inorganic compartments. In this respect, smart design, use of the appropriate organic ligands, and efficient and user-friendly synthesis of organometallic Rh complexes have played crucial roles in the inducing desirable biological activities. In this review, we focused on the recent advances published on the bioactivity of Rh (III/II/I) complexes especially inhibitory activity, from 2013 till now. Accordingly, considering the structure-activity relationship (SAR), the effect of oxidation state (+1, +2, and +3) and geometry (dimer or monomer complexes with coordination number of 4 and 6) of Rh complexes as well as various ligands on in vitro and in vivo studies was comprehensively discussed.


Assuntos
Complexos de Coordenação/química , Descoberta de Drogas , Ródio/química , Complexos de Coordenação/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Isomerases/antagonistas & inibidores , Isomerases/metabolismo , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade
11.
J Bacteriol ; 203(12): e0008621, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33782054

RESUMO

Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical S-adenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression. IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.


Assuntos
Proteínas de Transporte/metabolismo , Coenzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas Ferro-Enxofre/metabolismo , Isomerases/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Proteínas Ferro-Enxofre/genética , Isomerases/genética , Família Multigênica , Nitrato Redutase
12.
ACS Synth Biol ; 10(1): 158-172, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33395273

RESUMO

Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.


Assuntos
Saccharomyces cerevisiae/química , Sesquiterpenos/metabolismo , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Isomerases/genética , Isomerases/metabolismo , Magnésio/química , Engenharia Metabólica/métodos , Mutagênese Sítio-Dirigida , NADP/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/química
13.
J Biol Chem ; 296: 100031, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33154169

RESUMO

Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the ß-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into ß-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.


Assuntos
Isomerases/metabolismo , Ficobilinas/metabolismo , Ficoeritrina/metabolismo , Synechococcus/química , Urobilina/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias , Cromatografia Líquida , Isomerases/química , Isomerases/classificação , Biologia Marinha , Ficoeritrina/química , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/metabolismo , Synechococcus/genética , Espectrometria de Massas em Tandem , Urobilina/metabolismo
14.
J Biosci Bioeng ; 131(1): 47-52, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32994133

RESUMO

Aconitic acid, an unsaturated tricarboxylic acid, is used in the chemical industry as raw materials for organic synthesis, especially as a specific substrate for a flavoring agent. trans-Aconitic acid (tAA) is a trans-isomer of cis-aconitic acid and detected in some plants and bacteria. However, biosynthetic route and metabolism of tAA in relation to assimilation have been unknown. Aconitate isomerase (AI; EC 5.3.3.7) catalyzes the reversible isomerization between cis-aconitic acid and tAA. Pseudomonas sp. WU-0701 was isolated as a bacterium assimilating tAA as sole carbon source, and characterization and gene identification of AI were already reported. Here, we describe that Pseudomonas sp. WU-0701 exhibited growth in each synthetic medium containing glucose, citric acid, isocitric acid, or tAA as sole carbon source. AI was intracellularly detected all the time during the cultivation of the strain WU-0701 cells, irrespective of the carbon sources; AI activity was detected even in the glucose-grown cells. Through the subcellular fractionation experiments, AI was detected in the periplasmic fraction. This is the first report indicating that a bacterium belonging to the genus Pseudomonas is constitutive for the AI production.


Assuntos
Ácido Aconítico/metabolismo , Isomerases/biossíntese , Pseudomonas/metabolismo , Periplasma/metabolismo , Pseudomonas/citologia
15.
Chembiochem ; 22(1): 170-175, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-32790123

RESUMO

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature Tm (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in Tm value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes.


Assuntos
Isomerases/metabolismo , Temperatura , Estabilidade Enzimática , Isomerases/genética , Mutação , Engenharia de Proteínas
16.
J Plant Physiol ; 257: 153350, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33360493

RESUMO

Aconitate isomerase (EC 5.3.3.7) interconverts cis- and trans-isomers of aconitic acid. Expression of the gene encoding this enzyme was studied in maize (Zea mays L.) leaves depending on light regime. Aconitate isomerase was induced by white and by red light indicating the involvement of phytochrome in the regulation of gene expression. The enzyme was partially purified from maize leaves. The value of Km was 0.75 mM with cis-aconitate and 0.92 mM with trans-aconitate, pH optimum was 8.0-8.2 with both substrates, citrate and malate suppressed its activity. It is concluded that aconitate isomerase actively participates in the interconversion of cis- and trans-aconitate in the light providing a possibility of using the pool of trans-aconitate for the regulation of the tricarboxylic acid cycle activity and mediating citrate/isocitrate supply for the biosynthetic and signaling purposes in photosynthetic cells.


Assuntos
Expressão Gênica/efeitos da radiação , Isomerases/genética , Luz , Proteínas de Plantas/genética , Zea mays/genética , Ácido Aconítico/química , Isomerases/química , Isomerases/metabolismo , Cinética , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Zea mays/química , Zea mays/metabolismo
17.
PLoS One ; 15(12): e0243620, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33284858

RESUMO

Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.


Assuntos
Vias Biossintéticas , Bryopsida/metabolismo , Gotículas Lipídicas/metabolismo , Sesquiterpenos/metabolismo , Bryopsida/enzimologia , Isomerases/metabolismo , Proteínas de Plantas/metabolismo
18.
Microb Cell Fact ; 19(1): 200, 2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33138820

RESUMO

BACKGROUND: Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. RESULTS: Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. CONCLUSIONS: The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.


Assuntos
Alcenos/metabolismo , Diterpenos/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Antineoplásicos/metabolismo , Reatores Biológicos , Escherichia coli/metabolismo , Isomerases/metabolismo , Saccharomyces cerevisiae/genética , Solubilidade , Temperatura
19.
Mar Drugs ; 18(11)2020 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-33233366

RESUMO

The class EC 5.xx, a group of enzymes that interconvert optical, geometric, or positional isomers are interesting biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. This class, named "isomerases," can transform cheap biomolecules into expensive isomers with suitable stereochemistry useful in synthetic medicinal chemistry, and interesting cases of production of l-ribose, d-psicose, lactulose, and d-phenylalanine are known. However, in two published reports about potential biocatalysts of marine origin, isomerases are hardly mentioned. Therefore, it is of interest to deepen the knowledge of these biocatalysts from the marine environment with this specialized in-depth analysis conducted using a literature search without time limit constraints. In this review, the focus is dedicated mainly to example applications in biocatalysis that are not numerous confirming the general view previously reported. However, from this overall literature analysis, curiosity-driven scientific interest for marine isomerases seems to have been long-standing. However, the major fields in which application examples are framed are placed at the cutting edge of current biotechnological development. Since these enzymes can offer properties of industrial interest, this will act as a promoter for future studies of marine-originating isomerases in applied biocatalysis.


Assuntos
Organismos Aquáticos/enzimologia , Biotecnologia , Isomerases/metabolismo , Aminoácidos/metabolismo , Animais , Biocatálise , Humanos , Isomerases/isolamento & purificação , Metabolismo dos Lipídeos , Peptídeos/metabolismo , Estereoisomerismo , Especificidade por Substrato , Açúcares/metabolismo
20.
Int J Mol Sci ; 21(22)2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33238433

RESUMO

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-ß-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN--denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.


Assuntos
Plaquetas/metabolismo , Inflamação/genética , Agregação Plaquetária/genética , Trombospondina 1/sangue , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fibrinolisina/metabolismo , Humanos , Inflamação/sangue , Inflamação/metabolismo , Isomerases/genética , Isomerases/metabolismo , Selectina-P/sangue , Selectina-P/genética , Peptídeos/genética , Peptídeos/farmacologia , Plasminogênio/genética , Plasminogênio/metabolismo , Ativação Plaquetária/genética , Agregados Proteicos/genética , Conformação Proteica em Folha beta , Dobramento de Proteína/efeitos dos fármacos , Compostos de Sulfidrila/sangue , Compostos de Sulfidrila/metabolismo , Trombospondina 1/genética
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