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1.
Chembiochem ; 24(17): e202300290, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37167138

RESUMO

Alcohol dehydrogenases (ADH) are important tools for generating chiral α-hydroxyketones. Previously, only the ADH of Thauera aromatica was known to convert cyclic α-diketones with appropriate preference. Here, we extend the spectrum of suitable enzymes by three alcohol dehydrogenases from Citrifermentans bemidjiense (CibADH), Deferrisoma camini (DecADH), and Thauera phenylacetica (ThpADH). Of these, DecADH is characterized by very high thermostability; CibADH and ThpADH convert α-halogenated cyclohexanones with increased activity. Otherwise, however, the substrate spectrum of all four ADHs is highly conserved. Structural considerations led to the conclusion that conversion of diketones requires not only the expansion of the active site into a large binding pocket, but also the circumferential modification of almost all amino acid residues that form the first shell of the binding pocket. The constellation appears to be overall highly specific for the relative positioning of the carbonyl functions and the size of the C-ring.


Assuntos
Álcool Desidrogenase , Zinco , Sequência de Aminoácidos , Zinco/química , Álcool Desidrogenase/metabolismo , Domínio Catalítico , Aminoácidos , Cetonas
2.
Biochemistry ; 61(15): 1633-1641, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35856337

RESUMO

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.


Assuntos
Ribonucleotídeo Redutases , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Modelos Moleculares , Ribonucleotídeo Redutases/química
3.
J Biol Chem ; 297(2): 101008, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314684

RESUMO

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.


Assuntos
Evolução Molecular , Lactobacillus leichmannii/enzimologia , Fosfatos/química , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Sequência Conservada , Lactobacillus leichmannii/química , Fosfatos/metabolismo , Filogenia , Ligação Proteica , Especificidade por Substrato , Thermotoga maritima/química
4.
Chembiochem ; 23(3): e202100596, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-34859954

RESUMO

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Ribonucleotídeos/biossíntese , Acinetobacter/enzimologia , Aminoimidazol Carboxamida/química , Bactérias/enzimologia , Escherichia coli/enzimologia , Concentração de Íons de Hidrogênio , Polifosfatos/química , Ribonucleotídeos/química , Temperatura
5.
Chembiochem ; 23(15): e202200149, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35557486

RESUMO

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.


Assuntos
Álcool Desidrogenase , Thauera , Álcool Desidrogenase/química , Catálise , Cetonas/química , Especificidade por Substrato , Thauera/metabolismo , Zinco
6.
Int J Mol Sci ; 23(21)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36362425

RESUMO

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2'-deoxyribosyltransferase from Leishmania mexicana (LmPDT) and Escherichia coli hypoxanthine phosphoribosyltransferase (EcHPRT) were immobilized onto Ni2+-prechelated magnetic microspheres (MagReSyn®NTA). Among the resulting derivatives, MLmPDT3 (activity: 11,935 IU/gsupport, 63% retained activity, operational conditions: 40 °C and pH 5-7) and MEcHPRT3 (12,840 IU/gsupport, 45% retained activity, operational conditions: pH 5-8 and 40-60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the MLmPDT3/MEcHPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to MLmPDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, MLmPDT3/MEcHPRT3 was reused for 16 cycles, which displayed a 75% retained activity.


Assuntos
Cladribina , Enzimas Imobilizadas , Enzimas Imobilizadas/química , Pentosiltransferases , Magnetismo , Escherichia coli , Fenômenos Magnéticos
7.
Appl Environ Microbiol ; 86(14)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414796

RESUMO

Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an "orphan" ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αß defensins, and it was therefore renamed DerAB for defensin resistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell's potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell.IMPORTANCE Antimicrobial peptides (AMPs) play an important role in suppressing the growth of microorganisms. They can be produced by bacteria themselves-to inhibit competitors-but are also widely distributed in higher eukaryotes, including insects and mammals, where they form an important component of innate immunity. In low-GC-content Gram-positive bacteria, BceAB-like transporters play a crucial role in AMP resistance but have so far been primarily associated with interbacterial competition. Here, we show that the orphan transporter DerAB from the lactic acid bacterium Lactobacillus casei is crucial for high-level resistance against insect-derived AMPs. It therefore represents an important mechanism for interkingdom defense. Furthermore, our results support a signaling interference from DerAB on the PsdRSAB module that might prevent the activation of a full nisin response. The Bce modules from L. casei BL23 illustrate a biological paradox in which the intrinsic nisin detoxification potential only arises in the absence of a defensin-specific ABC transporter.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibiose , Proteínas de Bactérias/genética , Defensinas/química , Proteínas de Insetos/química , Lacticaseibacillus casei/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Lacticaseibacillus casei/metabolismo
8.
Sensors (Basel) ; 20(1)2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31905650

RESUMO

Bacterial biohybrid microswimmers aim at exploiting the inherent motion capabilities of bacteria (carriers) to transport objects (cargoes) at the microscale. One of the most desired properties of microswimmers is their ability to communicate with their immediate environment by processing the information and producing a useful response. Indeed, bacteria are naturally equipped with such communication skills. Hereby, two-component systems (TCSs) represent the key signal transducing machinery and enable bacteria to sense and respond to a variety of stimuli. We engineered a natural microswimmer based on the Gram-positive model bacterium Bacillus subtilis for the development of biohybrids with sensing abilities. B. subtilis naturally adhered to silica particles, giving rise to different motile biohybrids systems with variable ratios of carrier(s)-to-cargo(es). Genetically engineered TCS pathways allowed us to couple the binding to the inert particles with signaling the presence of antibiotics in their surroundings. Activation of the antibiotic-induced TCSs resulted in fluorescent bacterial carriers as a response readout. We demonstrate that the genetically engineered TCS-mediated signaling capabilities of B. subtilis allow for the custom design of bacterial hybrid microswimmers able to sense and signal the presence of target molecules in the environment. The generally recognized as safe (GRAS) status of B. subtilis makes it a promising candidate for human-related applications of these novel biohybrids.


Assuntos
Bacillus subtilis/genética , Técnicas Biossensoriais , Engenharia Genética , Bacillus subtilis/metabolismo , Transdução de Sinais , Dióxido de Silício/química
9.
J Biol Chem ; 292(46): 19044-19054, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-28972190

RESUMO

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.


Assuntos
Actinomycetales/química , Proteínas de Bactérias/química , Cisteína/química , Ribonucleotídeo Redutases/química , Dedos de Zinco , Actinomycetales/genética , Actinomycetales/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , Transporte de Elétrons , Modelos Moleculares , Oxirredução , Filogenia , Domínios Proteicos , Multimerização Proteica , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
10.
Chembiochem ; 16(10): 1512-9, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-26096455

RESUMO

Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4'-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the kcat ⋅KM (-1) value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.


Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Candida/enzimologia , Zinco/metabolismo , Acetofenonas/química , Acetofenonas/metabolismo , Álcool Desidrogenase/genética , Candida/química , Candida/genética , Candida/metabolismo , Domínio Catalítico , Análise Mutacional de DNA , Simulação de Acoplamento Molecular , Mutação Puntual , Conformação Proteica , Especificidade por Substrato
11.
J Comput Aided Mol Des ; 29(11): 1057-69, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26530855

RESUMO

Molecular docking of substrates is more challenging compared to inhibitors as the reaction mechanism has to be considered. This becomes more pronounced for zinc-dependent enzymes since the coordination state of the catalytic zinc ion is of greater importance. In order to develop a predictive substrate docking protocol, we have performed molecular docking studies of diketone substrates using the catalytic state of carbonyl reductase 2 from Candida parapsilosis (CPCR2). Different docking protocols using two docking methods (AutoDock Vina and AutoDock4.2) with two different sets of atomic charges (AM1-BCC and HF-RESP) for catalytic zinc environment and substrates as well as two sets of vdW parameters for zinc ion were examined. We have selected the catalytic binding pose of each substrate by applying mechanism based distance criteria. To compare the performance of the docking protocols, the correlation plots for the binding energies of these catalytic poses were obtained against experimental Vmax values of the 11 diketone substrates for CPCR2. The best correlation of 0.73 was achieved with AutoDock4.2 while treating catalytic zinc ion in optimized non-bonded (NBopt) state with +1.01 charge on the zinc ion, compared to 0.36 in non-bonded (+2.00 charge on the zinc ion) state. These results indicate the importance of catalytic constraints and charge parameterization of catalytic zinc environment for the prediction of substrate activity in zinc-dependent enzymes by molecular docking. The developed predictive docking protocol described here is in principle generally applicable for the efficient in silico substrate spectra characterization of zinc-dependent ADH.


Assuntos
Oxirredutases do Álcool/química , Simulação de Acoplamento Molecular , Conformação Proteica , Zinco/química , Oxirredutases do Álcool/metabolismo , Sítios de Ligação , Candida/enzimologia , Catálise , NAD/química , NAD/metabolismo , Ligação Proteica , Especificidade por Substrato
12.
PLoS One ; 17(8): e0268683, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35980975

RESUMO

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 µM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages.


Assuntos
Fabaceae , Cisteína/metabolismo , Defensinas/genética , Defensinas/metabolismo , Escherichia coli/metabolismo , Fabaceae/genética , Fabaceae/metabolismo , Regulação da Expressão Gênica de Plantas , Fixação de Nitrogênio , Peptídeos/metabolismo , Filogenia , Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Simbiose/genética
13.
Appl Environ Microbiol ; 77(15): 5483-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21685171

RESUMO

Spirochaeta thermophila is a thermophilic, free-living, and cellulolytic anaerobe. The genome sequence data for this organism have revealed a high density of genes encoding enzymes from more than 30 glycoside hydrolase (GH) families and a noncellulosomal enzyme system for (hemi)cellulose degradation. Functional screening of a fosmid library whose inserts were mapped on the S. thermophila genome sequence allowed the functional annotation of numerous GH open reading frames (ORFs). Seven different GH ORFs from the S. thermophila DSM 6192 genome, all putative ß-glycanase ORFs according to sequence similarity analysis, contained a highly conserved novel GH-associated module of unknown function at their C terminus. Four of these GH enzymes were experimentally verified as xylanase, ß-glucanase, ß-glucanase/carboxymethylcellulase (CMCase), and CMCase. Binding experiments performed with the recombinantly expressed and purified GH-associated module showed that it represents a new carbohydrate-binding module (CBM) that binds to microcrystalline cellulose and is highly specific for this substrate. In the course of this work, the new CBM type was only detected in Spirochaeta, but recently we found sequences with detectable similarity to the module in the draft genomes of Cytophaga fermentans and Mahella australiensis, both of which are phylogenetically very distant from S. thermophila and noncellulolytic, yet inhabit similar environments. This suggests a possibly broad distribution of the module in nature.


Assuntos
Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Spirochaeta/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Metabolismo dos Carboidratos , Carboidratos , Celulase/genética , Celulose/metabolismo , Cytophaga/genética , Cytophaga/metabolismo , Endo-1,4-beta-Xilanases/genética , Genoma Bacteriano , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Filogenia , Ligação Proteica/genética , Proteoglicanas/metabolismo , Receptores de Superfície Celular/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Spirochaeta/genética
14.
Biomolecules ; 11(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668847

RESUMO

Cladribine triphosphate is the active compound of the anti-cancer and multiple sclerosis drug Mavenclad (cladribine). Biosynthesis of such non-natural deoxyribonucleotides is challenging but important in order to study the pharmaceutical modes of action. In this study, we developed a novel one-pot enzyme cascade for the biosynthesis of cladribine triphosphate, starting with the nucleobase 2Cl-adenine and the generic co-substrate phosphoribosyl pyrophosphate. The cascade is comprised of the three enzymes, namely, adenine phosphoribosyltransferase (APT), polyphosphate kinase (PPK), and ribonucleotide reductase (RNR). APT catalyzes the binding of the nucleobase to the ribose moiety, followed by two consecutive phosphorylation reactions by PPK. The formed nucleoside triphosphate is reduced to the final product 2Cl-deoxyadenonsine triphosphate (cladribine triphosphate) by the RNR. The cascade is feasible, showing comparative product concentrations and yields to existing enzyme cascades for nucleotide biosynthesis. While this study is limited to the biosynthesis of cladribine triphosphate, the design of the cascade offers the potential to extend its application to other important deoxyribonucleotides.


Assuntos
Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ribonucleotídeo Redutases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Cladribina/análogos & derivados , Cladribina/metabolismo , Humanos , Nucleotídeos/metabolismo
15.
New Phytol ; 187(3): 751-63, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20609115

RESUMO

*At the end of the cell cycle, the plant cell wall is deposited within a membrane compartment referred to as the cell plate. Little is known about the biogenesis of this transient membrane compartment. *We have positionally cloned and characterized a novel Arabidopsis gene, CLUB, identified by mutation. *CLUB/AtTRS130 encodes a putative TRAPPII tethering factor. club mutants are seedling-lethal and have a canonical cytokinesis-defective phenotype, characterized by the appearance of bi- or multinucleate cells with cell wall stubs, gaps and floating walls. Confocal microscopy showed that in club mutants, KNOLLE-positive vesicles formed and accumulated at the cell equator throughout cytokinesis, but failed to assemble into a cell plate. Similarly, electron micrographs showed large vesicles loosely connected as patchy, incomplete cell plates in club root tips. Neither the formation of KNOLLE-positive vesicles nor the delivery of these vesicles to the cell equator appeared to be perturbed in club mutants. Thus, the primary defect in club mutants appears to be an impairment in cell plate assembly. *As a putative tethering factor required for cell plate biogenesis, CLUB/AtTRS130 helps to define the identity of this membrane compartment and comprises an important handle on the regulation of cell plate assembly.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Citocinese , Alelos , Arabidopsis/embriologia , Arabidopsis/ultraestrutura , Sequência Conservada , Meristema/citologia , Meristema/metabolismo , Meristema/ultraestrutura , Complexos Multiproteicos/metabolismo , Mutagênese Insercional/genética , Mutação/genética , Fenótipo , Pólen/metabolismo , Subunidades Proteicas/metabolismo , Plântula/metabolismo , Sementes/citologia , Sementes/metabolismo , Sementes/ultraestrutura
16.
PeerJ ; 7: e6700, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30993041

RESUMO

Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced Thermus spp. genome. Phylogenetic analysis revealed a distant origin of the nrdJm gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GC-content, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B12-dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60-70 °C, which is in the range of the growth optimum of Thermus spp. Experiments in combination with the Thermus thermophilus thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence.

17.
Elife ; 72018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29388911

RESUMO

Ribonucleotide reductases (RNRs) are key enzymes in DNA metabolism, with allosteric mechanisms controlling substrate specificity and overall activity. In RNRs, the activity master-switch, the ATP-cone, has been found exclusively in the catalytic subunit. In two class I RNR subclasses whose catalytic subunit lacks the ATP-cone, we discovered ATP-cones in the radical-generating subunit. The ATP-cone in the Leeuwenhoekiella blandensis radical-generating subunit regulates activity via quaternary structure induced by binding of nucleotides. ATP induces enzymatically competent dimers, whereas dATP induces non-productive tetramers, resulting in different holoenzymes. The tetramer forms by interactions between ATP-cones, shown by a 2.45 Å crystal structure. We also present evidence for an MnIIIMnIV metal center. In summary, lack of an ATP-cone domain in the catalytic subunit was compensated by transfer of the domain to the radical-generating subunit. To our knowledge, this represents the first observation of transfer of an allosteric domain between components of the same enzyme complex.


Assuntos
Trifosfato de Adenosina/metabolismo , Flavobacteriaceae/enzimologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Conformação Proteica , Multimerização Proteica
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