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1.
Appl Microbiol Biotechnol ; 108(1): 410, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38976076

RESUMO

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.


Assuntos
Oxirredutases do Álcool , Álcool Benzílico , Coenzimas , NADP , Oxirredução , Zinco , Concentração de Íons de Hidrogênio , NADP/metabolismo , Especificidade por Substrato , Álcool Benzílico/metabolismo , Álcool Benzílico/química , Cinética , Zinco/metabolismo , Coenzimas/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , NAD/metabolismo , Benzaldeídos/metabolismo , Benzaldeídos/química , Domínio Catalítico , Sítios de Ligação , Filogenia , Modelos Moleculares
2.
Appl Microbiol Biotechnol ; 108(1): 391, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38910188

RESUMO

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.


Assuntos
Coenzimas , Metais , Metais/metabolismo , Coenzimas/metabolismo , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Catálise , Oxirredutases/metabolismo , Oxirredutases/química
3.
Appl Environ Microbiol ; 90(6): e0023324, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38727223

RESUMO

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.


Assuntos
Benzaldeídos , Ácidos Cumáricos , Dioxigenases , Benzaldeídos/metabolismo , Ácidos Cumáricos/metabolismo , Dioxigenases/metabolismo , Dioxigenases/genética , Engenharia Metabólica , Coenzimas/metabolismo , Engenharia de Proteínas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
4.
Sci Rep ; 14(1): 11165, 2024 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750092

RESUMO

Kinetic aspects of enzymatic reactions are described by equations based on the Michaelis-Menten theory for the initial stage. However, the kinetic parameters provide little information on the atomic mechanism of the reaction. In this study, we analyzed structures of glutamate dehydrogenase in the initial and steady stages of the reaction using cryoEM at near-atomic resolution. In the initial stage, four metastable conformations displayed different domain motions and cofactor/ligand association modes. The most striking finding was that the enzyme-cofactor-substrate complex, treated as a single state in the enzyme kinetic theory, comprised at least three different metastable conformations. In the steady stage, seven conformations, including derivatives from the four conformations in the initial stage, made the reaction pathway complicated. Based on the visualized conformations, we discussed stage-dependent pathways to illustrate the dynamics of the enzyme in action.


Assuntos
Microscopia Crioeletrônica , Glutamato Desidrogenase , Conformação Proteica , Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Microscopia Crioeletrônica/métodos , Ligantes , Cinética , Modelos Moleculares , Coenzimas/metabolismo , Coenzimas/química , Catálise , Ligação Proteica
5.
FEMS Yeast Res ; 242024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38637306

RESUMO

Anaerobic alcoholic fermentation, particularly in high-sugar environments, presents metabolic challenges for yeasts. Crabtree-positive yeasts, including Saccharomyces cerevisiae, prefer fermentation even in the presence of oxygen. These yeasts rely on internal NAD+ recycling and extracellular assimilation of its precursor, nicotinic acid (vitamin B3), rather than de novo NAD+ production. Surprisingly, nicotinic acid assimilation is poorly characterized, even in S. cerevisiae. This study elucidated the timing of nicotinic acid uptake during grape juice-like fermentation and its impact on NAD(H) levels, the NAD+/NADH ratio, and metabolites produced. Complete uptake of extracellular nicotinic acid occurred premid-exponential phase, thereafter small amounts of vitamin B3 were exported back into the medium. Suboptimal levels of nicotinic acid were correlated with slower fermentation and reduced biomass, disrupting redox balance and impeding NAD+ regeneration, thereby affecting metabolite production. Metabolic outcomes varied with nicotinic acid concentrations, linking NAD+ availability to fermentation efficiency. A model was proposed encompassing rapid nicotinic acid uptake, accumulation during cell proliferation, and recycling with limited vitamin B3 export. This research enhances the understanding of nicotinic acid uptake dynamics during grape juice-like fermentation. These insights contribute to advancing yeast metabolism research and have profound implications for the enhancement of biotechnological practices and the wine-making industry.


Assuntos
Fermentação , NAD , Niacina , Oxirredução , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Niacina/metabolismo , NAD/metabolismo , Etanol/metabolismo , Coenzimas/metabolismo
6.
Biochim Biophys Acta Mol Cell Res ; 1871(5): 119731, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38631442

RESUMO

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.


Assuntos
Coenzimas , Proteínas Ferro-Enxofre , Metaloproteínas , Cofatores de Molibdênio , Pteridinas , Metaloproteínas/metabolismo , Metaloproteínas/genética , Metaloproteínas/biossíntese , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/genética , Coenzimas/metabolismo , Coenzimas/biossíntese , Coenzimas/genética , Pteridinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ferro/metabolismo , Enxofre/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Isomerases
7.
Chemphyschem ; 25(14): e202400327, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38602444

RESUMO

The present work is another part of our investigation on the pathway of dissimilatory sulfate reduction and covers a theoretical study on the reaction catalyzed by dissimilatory sulfite reductase (dSIR). dSIR is the terminal enzyme involved in this metabolic pathway, which uses the siroheme-[4Fe4S] cofactor for six-electron reduction of sulfite to sulfide. In this study we use a large cluster model containing siroheme-[4Fe4S] cofactor and protein residues involved in the direct interactions with the substrate, to get insight into the most feasible reaction mechanism and to understand the role of each considered active site component. In combination with earlier studies reported in the literature, our results lead to several interesting insights. One of the most important conclusions is that the reaction mechanism consists of three steps of two-electron reduction of sulfur and the probable role of the siroheme-[4Fe4S] cofactor is to ensure the delivery of packages of two electrons to the reactant.


Assuntos
Heme , Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Heme/química , Heme/metabolismo , Heme/análogos & derivados , Biocatálise , Sulfito de Hidrogênio Redutase/metabolismo , Sulfito de Hidrogênio Redutase/química , Domínio Catalítico , Oxirredução , Sulfitos/química , Sulfitos/metabolismo , Coenzimas/metabolismo , Coenzimas/química , Modelos Moleculares
8.
Biotechnol J ; 19(4): e2300557, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38581092

RESUMO

The halogenase-based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin-dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site-selectivity and wide substrate range. However, the complexity of constructing the NAD+-NADH-FAD-FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self-sufficient tri-enzyme fusion was constructed and successfully applied to the continuous halogenation of L-tryptophan. SpFDH was firstly identified derived from Streptomyces pratensis, a highly selective halogenase capable of generating 6-chloro-tryptophan from tryptophan. Then, using gene fusion technology, SpFDH was fused with glucose dehydrogenase (GDH) and flavin reductase (FR) to form a tri-enzyme fusion, which increased the yield by 1.46-fold and making the coenzymes self-sufficient. For more efficient halogenation of L-tryptophan, a continuous halogenation bioprocess of L-tryptophan was developed by immobilizing the tri-enzyme fusion and attaching it to a continuous catalytic device, which resulted in a reaction yield of 97.6% after 12 h reaction. An FDH from S. pratensis was successfully applied in the halogenation and our study provides a concise strategy for the preparation of halogenated tryptophan mediated by multienzyme cascade catalysis.


Assuntos
Halogenação , Triptofano , Coenzimas , Oxirredutases/genética , Oxirredutases/metabolismo , Flavinas/metabolismo
9.
Genes (Basel) ; 15(3)2024 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-38540376

RESUMO

The marine nematode Litoditis marina is widely distributed in intertidal zones around the globe, yet the mechanisms underlying its broad adaptation to salinity remain elusive. In this study, we applied ONT long-read sequencing technology to unravel the transcriptome responses to different salinity conditions in L. marina. Through ONT sequencing under 3‱, 30‱ and 60‱ salinity environments, we obtained 131.78 G clean data and 26,647 non-redundant long-read transcripts, including 6464 novel transcripts. The DEGs obtained from the current ONT lrRNA-seq were highly correlated with those identified in our previously reported Illumina short-read RNA sequencing data. When we compared the 30‱ to the 3‱ salinity condition, we found that GO terms such as oxidoreductase activity, cation transmembrane transport and ion transmembrane transport were shared between the ONT lrRNA-seq and Illumina data. Similarly, GO terms including extracellular space, structural constituents of cuticle, substrate-specific channel activity, ion transport and substrate-specific transmembrane transporter activity were shared between the ONT and Illumina data under 60‱ compared to 30‱ salinity. In addition, we found that 79 genes significantly increased, while 119 genes significantly decreased, as the salinity increased. Furthermore, through the GO enrichment analysis of 214 genes containing DAS, in 30‱ compared to 3‱ salinity, we found that GO terms such as cellular component assembly and coenzyme biosynthetic process were enriched. Additionally, we observed that GO terms such as cellular component assembly and coenzyme biosynthetic process were also enriched in 60‱ compared to 30‱ salinity. Moreover, we found that 86, 125, and 81 genes that contained DAS were also DEGs, in comparisons between 30‱ and 3‱, 60‱ and 30‱, and 60‱ and 3‱ salinity, respectively. In addition, we demonstrated the landscape of alternative polyadenylation in marine nematode under different salinity conditions This report provides several novel insights for the further study of the mechanisms by which euryhalinity formed and evolved, and it might also contribute to the investigation of salinity dynamics induced by global climate change.


Assuntos
Salinidade , Transcriptoma , Transcriptoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Coenzimas
10.
J Biol Chem ; 300(3): 105729, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38336296

RESUMO

RNase P and RNase mitochondrial RNA processing (MRP) are ribonucleoproteins (RNPs) that consist of a catalytic RNA and a varying number of protein cofactors. RNase P is responsible for precursor tRNA maturation in all three domains of life, while RNase MRP, exclusive to eukaryotes, primarily functions in rRNA biogenesis. While eukaryotic RNase P is associated with more protein cofactors and has an RNA subunit with fewer auxiliary structural elements compared to its bacterial cousin, the double-anchor precursor tRNA recognition mechanism has remarkably been preserved during evolution. RNase MRP shares evolutionary and structural similarities with RNase P, preserving the catalytic core within the RNA moiety inherited from their common ancestor. By incorporating new protein cofactors and RNA elements, RNase MRP has established itself as a distinct RNP capable of processing ssRNA substrates. The structural information on RNase P and MRP helps build an evolutionary trajectory, depicting how emerging protein cofactors harmonize with the evolution of RNA to shape different functions for RNase P and MRP. Here, we outline the structural and functional relationship between RNase P and MRP to illustrate the coevolution of RNA and protein cofactors, a key driver for the extant, diverse RNP world.


Assuntos
Endorribonucleases , Evolução Molecular , Subunidades Proteicas , RNA Catalítico , Ribonuclease P , Coenzimas , Endorribonucleases/química , Endorribonucleases/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonuclease P/química , Ribonuclease P/metabolismo , Processamento Pós-Transcricional do RNA , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Especificidade por Substrato , Eucariotos/enzimologia
11.
ACS Nano ; 18(9): 7011-7023, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38390865

RESUMO

Ferroptotic cancer therapy has been extensively investigated since the genesis of the ferroptosis concept. However, the therapeutic efficacy of ferroptosis induction in heterogeneous and plastic melanoma has been compromised, because the melanocytic and transitory cell subpopulation is resistant to iron-dependent oxidative stress. Here, we report a phenotype-altering liposomal nanomedicine to enable the ferroptosis-resistant subtypes of melanoma cells vulnerable to lipid peroxidation via senescence induction. The strategy involves the ratiometric coencapsulation of a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor (palbociclib) and a ferroptosis inducer (auranofin) within cRGD peptide-modified targeted liposomes. The two drugs showed a synergistic anticancer effect in the model B16F10 melanoma cells, as evidenced by the combination index analysis (<1). The liposomes could efficiently deliver both drugs into B16F10 cells in a targeted manner. Afterward, the liposomes potently induced the intracellular redox imbalance and lipid peroxidation. Palbociclib significantly provoked cell cycle arrest at the G0/G1 phase, which sensitized auranofin-caused ferroptosis through senescence induction. Meanwhile, palbociclib depleted intracellular glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), further boosting ferroptosis. The proof-of-concept was also demonstrated in the B16F10 tumor-bearing mice model. The current work offers a promising ferroptosis-targeting strategy for effectively treating heterogeneous melanoma by manipulating the cellular plasticity.


Assuntos
Ferroptose , Melanoma , Animais , Camundongos , Melanoma/tratamento farmacológico , Lipossomos/farmacologia , Coenzimas/farmacologia , Auranofina/farmacologia , Peroxidação de Lipídeos
12.
Angew Chem Int Ed Engl ; 63(15): e202319966, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38327168

RESUMO

Albeit sonodynamic therapy (SDT) has achieved encouraging progress in microbial sterilization, the scarcity of guidelines for designing highly effective sonosensitizers and the intricate biofilm microenvironment (BME), substantially hamper the therapeutic efficacy against biofilm infections. To address the bottlenecks, we innovatively design a Ru(II) metallacycle-based sonosensitizer/sonocatalyst (named Ru-A3-TTD) to enhance the potency of sonotherapy by employing molecular engineering strategies tailored to BME. Our approach involves augmenting Ru-A3-TTD's production of ultrasonic-triggered reactive oxygen species (ROS), surpassing the performance of commercial sonosensitizers, through a straightforward but potent π-expansion approach. Within the BME, Ru-A3-TTD synergistically amplifies sonotherapeutic efficacy via triple-modulated approaches: (i) effective alleviation of hypoxia, leading to increased ROS generation, (ii) disruption of the antioxidant defense system, which shields ROS from glutathione consumption, and (iii) enhanced biofilm penetration, enabling ROS production in deep sites. Notably, Ru-A3-TTD sono-catalytically oxidizes NADPH, a critical coenzyme involved in antioxidant defenses. Consequently, Ru-A3-TTD demonstrates superior biofilm eradication potency against multidrug-resistant Escherichia coli compared to conventional clinical antibiotics, both in vitro and in vivo. To our knowledge, this study represents the pioneering instance of a supramolecular sonosensitizer/sonocatalyst. It provides valuable insights into the structure-activity relationship of sonosensitizers and paves a promising pathway for the treatment of biofilm infections.


Assuntos
Antioxidantes , Neoplasias , Humanos , Espécies Reativas de Oxigênio , Antibacterianos/farmacologia , Biofilmes , Coenzimas , Escherichia coli , Linhagem Celular Tumoral , Microambiente Tumoral
13.
J Phys Chem B ; 128(6): 1428-1437, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38301132

RESUMO

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.


Assuntos
Coenzimas , Vitamina B 12 , Espectroscopia por Absorção de Raios X , Vitamina B 12/química , Fotólise
14.
Biochim Biophys Acta Mol Basis Dis ; 1870(3): 167038, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38281710

RESUMO

Nicotinamide adenine dinucleotide (NAD) coenzymes are carriers of high energy electrons in metabolism and also play critical roles in numerous signaling pathways. NAD metabolism is decreased in various cardiovascular diseases. Importantly, stimulation of NAD biosynthesis protects against heart disease under different pathological conditions. In this review, we describe pathways for both generation and catabolism of NAD coenzymes and the respective changes of these pathways in the heart under cardiac diseases, including pressure overload, myocardial infarction, cardiometabolic disease, cancer treatment cardiotoxicity, and heart failure. We next provide an update on the strategies and treatments to increase NAD levels, such as supplementation of NAD precursors, in the heart that prevent or reverse cardiomyopathy. We also introduce the approaches to manipulate NAD consumption enzymes to ameliorate cardiac disease. Finally, we discuss the mechanisms associated with improvements in cardiac function by NAD coenzymes, differentiating between mitochondria-dependent effects and those independent of mitochondrial metabolism.


Assuntos
Cardiopatias , Insuficiência Cardíaca , Humanos , NAD/metabolismo , Remodelação Ventricular , Coração , Coenzimas
15.
Chem Biol Interact ; 390: 110876, 2024 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-38266864

RESUMO

The medium-chain dehydrogenase/reductase (MDR) superfamily has more than 600,000 members in UniProt as of March 2023. As the family has been growing, the proportion of functionally characterized proteins has been falling behind. The aim of this project was to investigate the binding pockets of nine different MDR protein families based on sequence conservation patterns and three-dimensional structures of members within the respective families. A search and analysis methodology was developed. Using this, a total of 2000 eukaryotic MDR sequences belonging to nine different families were identified. The pairwise sequence identities within each of the families were 80-90 % for the mammalian sequences, like the levels observed for alcohol dehydrogenase, another MDR family. Twenty conserved residues were identified in the coenzyme part of the binding site by matching structural and conservation data of all nine protein families. The conserved residues in the substrate part of the binding pocket varied between the nine MDR families, implying divergent functions for the different families. Studying each family separately made it possible to identify multiple conserved residues that are expected to be important for substrate binding or catalysis of the enzymatic reaction. By combining structural data with the conservation of the amino acid residues in each protein, important residues in the binding pocket were identified for each of the nine MDRs. The obtained results add new positions of interest for the binding and activity of the enzyme family as well as fit well to earlier published data. Three distinct types of binding pockets were identified, containing no, one, or two tyrosine residues.


Assuntos
Álcool Desidrogenase , Coenzimas , Animais , Humanos , Coenzimas/metabolismo , Álcool Desidrogenase/metabolismo , Mamíferos/metabolismo
16.
Neurosci Lett ; 821: 137623, 2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38184017

RESUMO

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.


Assuntos
Proteínas de Ligação a Calmodulina , Cloretos , Compostos Férricos , Proteínas do Tecido Nervoso , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Íons , Coenzimas/metabolismo
17.
Prep Biochem Biotechnol ; 54(1): 103-114, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37184437

RESUMO

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.


Assuntos
Quitosana , Glutamato Descarboxilase/química , Ácido gama-Aminobutírico , Coenzimas , Fenômenos Magnéticos
18.
Chembiochem ; 25(1): e202300409, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-37948327

RESUMO

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.


Assuntos
Coenzimas , Ferredoxinas , Coenzimas/metabolismo , Ferredoxinas/metabolismo , Oxirredutases/metabolismo , Escherichia coli/metabolismo , Oxirredução
19.
Cell Rep ; 42(12): 113571, 2023 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-38096053

RESUMO

Natural polyamines such as spermidine and spermine cations have characteristics that make them highly likely to be sensed by riboswitches, such as their general affinity to polyanionic RNA and their broad contributions to cell physiology. Despite previous claims that polyamine riboswitches exist, evidence of their biological functions has remained unconvincing. Here, we report that rare variants of bacterial S-adenosylmethionine-I (SAM-I) riboswitches reject SAM and have adapted to selectively sense spermidine. These spermidine-sensing riboswitch variants are associated with genes whose protein products are directly involved in the production of spermidine and other polyamines. Biochemical and genetic assays demonstrate that representatives of this riboswitch class robustly function as genetic "off" switches, wherein spermidine binding causes premature transcription termination to suppress the expression of polyamine biosynthetic genes. These findings confirm the existence of natural spermidine-sensing riboswitches in bacteria and expand the list of variant riboswitch classes that have adapted to bind different ligands.


Assuntos
Riboswitch , Riboswitch/genética , S-Adenosilmetionina/metabolismo , Espermidina , Coenzimas/metabolismo , Oligonucleotídeos , Bactérias/genética , Bactérias/metabolismo , Conformação de Ácido Nucleico
20.
J Agric Food Chem ; 71(51): 20772-20781, 2023 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-37963219

RESUMO

l-Phenyllactic acid (l-PLA) is a small molecular organic acid that exhibits a powerful capacity for inhibition against foodborne pathogens. In this work, we developed a new cost-effective and environmentally friendly process for the biosynthesis of l-PLA. This strategy designed a novel whole-cell biotransformation system employing two heterologous enzymes, namely, phenylalanine dehydrogenase (PheDH) and l-hydroxyisocaproate dehydrogenase (l-HicDH). The novelty of this strategy lies in the first-time utilization of these two enzymes, which not only enables cascade catalysis for the production of l-PLA but also facilitates the regeneration of the coenzymes (NAD+/NADH) using only two enzymes rather than introducing more heterologous enzymes to the system. Consequently, this strategy can effectively simplify the biosynthesis process of l-PLA and minimize production costs. The initial l-PLA yield using this process achieved 2.53 ± 0.07 g/L. Furthermore, through meticulous optimization of the parameters for inducible enzyme expression and l-PLA biosynthesis, the l-PLA yield was successfully increased to 4.68 ± 0.04 g/L with a yield rate of 64.54 ± 0.29%. Moreover, this novel strategy is versatile in the biosynthesis of other organic acids, which can be achieved by easily modulating the combinations of substrates and enzymes.


Assuntos
Coenzimas , Regeneração , Biotransformação , Poliésteres
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