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Abstract Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C–C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning–guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. Key points • DERA aldolases are versatile biocatalysts able to make new C–C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts. Graphical abstract

In nature 2-deoxy-D-ribose-5-phosphate aldolase (DERA) catalyses the reversible formation of 2-deoxyribose 5-phosphate from D-glyceraldehyde 3-phosphate and acetaldehyde. In addition, this enzyme can use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6-trideoxy-D-erythro-hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin-type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2-deoxyribose 5-phosphate and 2,4,6-trideoxy-D-erythro-hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C-His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole-cell experiment which makes it suitable for industrial applications.

Analogues of the Ca2+-releasing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [1, Ins(1,4,5)P3] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d-chiro-inositol in d-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P3 mimic ribophostin 6 may increase the activity. d-chiro-Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with l-(+)-3-O-trifluoromethylsulfonyl-6-O-p-methoxybenzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 11. Separable diastereoisomeric 3-O-camphanate esters of (±)-6-O-p-methoxy-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol allowed the preparation of 11. Selective trans-isopropylidene deprotection in coupled 13, then monobenzylation gave separable regioisomers 15 and 16. p-Methoxybenzyl group deprotection of 16, phosphitylation/oxidation, then deprotection afforded 10, which was a full agonist in Ca2+-release assays; its potency and binding affinity for Ins(1,4,5)P3R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca2+ current ICRAC in patch-clamped cells, unlike Ins(1,4,5)P3 consistent with resistance to metabolism. d-chiro-Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P3 receptor agonist without a nucleobase yet synthesized.

A simple and efficient method for the preparation of α-D-ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate, key intermediates in nucleoside metabolism and important starting compounds for the enzymatic synthesis of various modified nucleosides, has been proposed. It consists in near-irreversible enzymatic phosphorolysis of readily prepared hydroiodide salts of 7-methylguanosine and 7-methyl-2'-deoxyguanosine, respectively, in the presence of purine nucleoside phosphorylase. α-D-Ribose 1-phosphate and 2-deoxy-α-D-ribose 1-phosphate are obtained in near quantitative yields (by HPLC analysis) and 74%-94% yields after their isolation and purification. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of α-D-ribose 1-phosphate barium salt (4a) Alternate Protocol 1: Preparation of 2-deoxy-α-D-ribose 1-phosphate barium salt (4b) Basic Protocol 2: Preparation of α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5a) Alternate Protocol 2: Preparation of 2-deoxy-α-D-ribose 1-phosphate bis(cyclohexylammonium) salt (5b).

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network., The evolutionary origins of metabolism are largely unknown. This study shows that the prebiotically available proteinogenic amino acid cysteine can promote the metabolism-like rate-limiting formation of ribose-5-phosphate, suggesting that early metabolic pathways could have emerged thought the stepwise enzymatization of non-enzymatic reaction sequences.

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O2 availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-3H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following 3 weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-3H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH up-regulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase muscle isoenzyme M2 (PKM2) protein content 1.4-fold compared with normoxic hearts. CH also augmented cardiac pentose phosphate pathway (PPP) flux, reflected by higher ribose-5-phosphate (R5P) content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained up-regulation of the glycolytic flux in the chronically hypoxic heart.

Anterograde and retrograde tract tracing were combined with neurotransmitter and modulator immunolabeling to identify the chemical anatomy of vestibular nuclear neurons with direct projections to the solitary nucleus in rats. Direct, sparsely branched but highly varicose axonal projections from neurons in the caudal vestibular nuclei to the solitary nucleus were observed. The vestibular neurons giving rise to these projections were predominantly located in ipsilateral medial vestibular nucleus. The cell bodies were intensely glutamate immunofluorescent, and their axonal processes contained vesicular glutamate transporter 2, supporting the interpretation that the cells utilize glutamate for neurotransmission. The glutamate-immunofluorescent, retrogradely filled vestibular cells also contained the neuromodulator imidazoleacetic acid ribotide, which is an endogenous CNS ligand that participates in blood pressure regulation. The vestibulo-solitary neurons were encapsulated by axo-somatic GABAergic terminals, suggesting that they are under tight inhibitory control. The results establish a chemoanatomical basis for transient vestibular activation of the output pathways from the caudal and intermediate regions of the solitary nucleus. In this way, changes in static head position and movement of the head in space may directly influence heart rate, blood pressure, respiration, as well as gastrointestinal motility. This would provide one anatomical explanation for the synchronous heart rate and blood pressure responses observed after peripheral vestibular activation, as well as disorders ranging from neurogenic orthostatic hypotension, postural orthostatic tachycardia syndrome, and vasovagal syncope to the nausea and vomiting associated with motion sickness. NEW & NOTEWORTHY Vestibular neurons with direct projections to the solitary nucleus utilize glutamate for neurotransmission, modulated by imidazoleacetic acid ribotide. This is the first direct demonstration of the chemical neuroanatomy of the vestibulo-solitary pathway.

Thymidine phosphorylase (TP; EC 2.4.2.4) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective nucleobases and 2-deoxy-α-d-ribose-1-phosphate (dRib-1-P). TP is a key enzyme in the pyrimidine salvage pathways. Activity of the enzyme is crucial in angiogenesis, cancer chemotherapy, radiotherapy, and tumor imaging, Nevertheless, a complete set of kinetic parameters has never been reported for any human TP. This study describes the kinetic mechanism and regulation of native human hepatic TP. The liver is a main site of pyrimidine metabolism and contains high levels of TP. Initial velocity and product inhibition studies demonstrated that the basic mechanism of this enzyme is a sequential random bi-bi mechanism. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation, and a binding pattern indicating that the binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KThymidine = 284 ± 55, KPi = 5.8 ± 1.9, KThymine = 244 ± 69, and KdRib-1-P = 90 ± 33 μM. Thymine was a product activator, but becomes a substrate inhibitor at concentrations eight times higher than its Km. dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bounded better to the Enzyme●Pi complex than the free enzyme, but had better affinity to the free enzyme than the Enzyme●Thymidine complex. In the reverse reaction, dRib-1-P enhanced the binding of thymine. The enhancement of the thymine binding along with the fact that dRib-1-P was a non-competitive product inhibitor suggests the presence of another binding site for dRib-1-P on the enzyme.

6-Phosphogluconate dehydrogenase (6PGD) is a key enzyme that converts 6-phosphogluconate into ribulose-5-phosphate with NADP+ as cofactor in the pentose phosphate pathway (PPP). 6PGD is commonly upregulated and plays important roles in many human cancers, while the mechanism underlying such roles of 6PGD remains elusive. Here we show that upon EGFR activation, 6PGD is phosphorylated at tyrosine (Y) 481 by Src family kinase Fyn. This phosphorylation enhances 6PGD activity by increasing its binding affinity to NADP+ and therefore activates the PPP for NADPH and ribose-5-phosphate, which consequently detoxifies intracellular reactive oxygen species (ROS) and accelerates DNA synthesis. Abrogating 6PGD Y481 phosphorylation (pY481) dramatically attenuates EGF-promoted glioma cell proliferation, tumor growth and resistance to ionizing radiation. In addition, 6PGD pY481 is associated with Fyn expression, the malignancy and prognosis of human glioblastoma. These findings establish a critical role of Fyn-dependent 6PGD phosphorylation in EGF-promoted tumor growth and radiation resistance., 6-phosphogluconate dehydrogenase is commonly upregulated in cancers. Here, the authors show that activation of EGFR induces phosphorylation of this enzyme at Y481 to activate the pentose phosphate pathway, which consequently reduces ROS and accelerates DNA synthesis to promote tumor growth and radioresistance.

Funding from the European Community's Seventh Framework Programme under grant agreements No. 602773 (Project KINDRED) was received for all partners in this work. This work also received funds from FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior through the Research Unit No. 4293 and project POCI-01-0145-FEDER-031013 (PTDC/SAUPAR/31013/2017 to NS); Individual funding from FCT through SFRH/BD/133485/2017 (to MS) and CEECIND/02362/2017 (to JT). Neglected tropical diseases caused by kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp.) place a significant health and economic burden on developing nations worldwide. Current therapies are largely out-dated, inadequate and facing mounting drug resistance from the causative parasites. Thus, there is an urgent need for drug discovery and development. Target-led drug discovery approaches have focused on the identification of parasite enzymes catalysing essential biochemical processes, which significantly differ from equivalent proteins found in humans, thereby providing potentially exploitable therapeutic windows. One such target is ribose 5-phosphate isomerase B (RpiB), an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, which catalyses the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Although protozoan RpiB has been the focus of numerous targeted studies, compounds capable of selectively inhibiting this parasite enzyme have not been identified. Here, we present the results of a fragment library screening against Leishmania infantum RpiB, performed using thermal shift analysis. Hit fragments were shown to be effective inhibitors of LiRpiB in activity assays, and several were capable of selectively inhibiting parasite growth in vitro. These results support the identification of LiRpiB as a validated therapeutic target. The X-ray crystal structure of apo LiRpiB was also solved, permitting docking studies to assess how hit fragments might interact with LiRpiB to inhibit its activity. Overall, this work will guide structure-based development of LiRpiB inhibitors as anti-leishmanial agents. Postprint

The Fibro-purF motif is a putative structured noncoding RNA domain that was discovered previously in species of Fibrobacter by using comparative sequence analysis methods. An updated bioinformatics search yielded a total of only 30 unique-sequence representatives, exclusively found upstream of the purF gene that codes for the enzyme amidophosphoribosyltransferase. This enzyme synthesizes the compound 5-phospho-D-ribosylamine (PRA), which is the first committed step in purine biosynthesis. The consensus model for Fibro-purF motif RNAs includes a predicted three-stem junction that carries numerous conserved nucleotide positions within the regions joining the stems. This architecture appears to be of sufficient size and complexity for the formation of the ligand-binding aptamer portion of a riboswitch. In this study, we conducted biochemical analyses of a representative Fibro-purF motif RNA to confirm that the RNA generally folds according to the predicted consensus model. However, due to the instability of PRA, binding of this ligand candidate by the RNA could not be directly assessed. Genetic analyses were used to demonstrate that Fibro-purF motif RNAs regulate gene expression in accordance with predicted PRA concentrations. These findings indicate that Fibro-purF motif RNAs are genetic regulation elements that likely suppress PRA biosynthesis when sufficient levels of this purine precursor are present.

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3′-phospho-α,β-unsaturated aldehyde (PUA) and 5′-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3′-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5′-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5′-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.

Uridine phosphorylase (UP) is a key enzyme of pyrimidine salvage pathways that enables the recycling of endogenous or exogenous-supplied pyrimidines and plays an important intracellular metabolic role. Here, we biochemically and structurally characterized two evolutionarily divergent uridine phosphorylases, PcUP1 and PcUP2 from the oomycete pathogen Phytophthora capsici. Our analysis of other oomycete genomes revealed that both uridine phosphorylases are present in Phytophthora and Pythium genomes, but only UP2 is seen in Saprolegnia spp. which are basal members of the oomycetes. Moreover, uridine phosphorylases are not found in obligate oomycete pathogens such as Hyaloperonospora arabidopsidis and Albugo spp. PcUP1 and PcUP2 are upregulated 300 and 500 fold respectively, within 90 min after infection of pepper leaves. The crystal structures of PcUP1 in ligand-free and in complex with uracil/ribose-1-phosphate, 2′-deoxyuridine/phosphate and thymidine/phosphate were analyzed. Crystal structure of this uridine phosphorylase showed strict conservation of key residues in the binding pocket. Structure analysis of PcUP1 with bound ligands, and site-directed mutagenesis of key residues provide additional support for the “push-pull” model of catalysis. Our study highlights the importance of pyrimidine salvage during the earliest stages of infection.

Ribose-5-phosphate isomerase (Rpi, EC 5.3.1.6) is widespread in microorganisms, animals, and plants. It has a pivotal role in the pentose phosphate pathway and responsible for catalyzing the isomerization between D-ribulose 5-phosphate and D-ribose 5-phosphate. In recent years, Rpi has received considerable attention as a multipurpose biocatalyst for production of rare sugars, including D-allose, L-rhamnulose, L-lyxose, and L-tagatose. Besides, it has been thought of as a potential drug target in the treatment of trypanosomatid-caused diseases such as Chagas' disease, leishmaniasis, and human African trypanosomiasis. Despite increased research activities, up to now, no systematic review of Rpi has been published. To fill this gap, this paper provides detailed information about the enzymatic properties of various Rpis. Furthermore, structural features, catalytic mechanism, and molecular modifications of Rpis are summarized based on extensive crystal structure research. Additionally, the applications of Rpi in rare sugar production and the role of Rpi in trypanocidal drug design are reviewed. Key points • Fundamental properties of various ribose-5-phosphate isomerases (Rpis). • Differences in crystal structure and catalytic mechanism between RpiA and RpiB. • Application of Rpi as a rare sugar producer and a potential drug target.

Summary Methanol, being electron rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can efficiently utilize methanol as the sole carbon source. This synthetic methylotroph alleviated a so far uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS)-mediated copy number variations (CNVs) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable with natural methylotrophs in a wide range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes and expands the scope of biological C1 conversion.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils – potentially important in the microenvironment of actively dividing cells, such as cancer cells – disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5′ and 3′ ends meet by diffusion-controlled encounter “through solution” or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5′–3′ interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3′ UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5′–3′ polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5′ end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3′ UTR and the 5′ end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap., RNA, 26 (5), ISSN:1355-8382, ISSN:1469-9001

Treatments on neoplastic diseases and cancer using genotoxic drugs often cause long-term health problems related to premature aging. The underlying mechanism is poorly understood. Based on the study of a long-lasting senescence-like growth arrest (10-12 weeks) of human dermal fibroblasts induced by psoralen plus UVA (PUVA) treatment, we here revealed that slowly repaired bulky DNA damages can serve as a “molecular scar” leading to reduced cell proliferation through persistent endogenous production of reactive oxygen species (ROS) that caused accelerated telomere erosion. The elevated levels of ROS were the results of mitochondrial dysfunction and the activation of NADPH oxidase (NOX). A combined inhibition of DNA-PK and PARP1 could suppress the level of ROS. Together with a reduced expression level of BRCA1 as well as the upregulation of PP2A and 53BP1, these data suggest that the NHEJ repair of DNA double-strand breaks may be the initial trigger of metabolic changes leading to ROS production. Further study showed that stimulation of the pentose phosphate pathway played an important role for NOX activation, and ROS could be efficiently suppressed by modulating the NADP/NADPH ratio. Interestingly, feeding cells with ribose-5-phosphate, a precursor for nucleotide biosynthesis that produced through the PPP, could evidently suppress the ROS level and prevent the cell enlargement related to mitochondrial biogenesis. Taken together, these results revealed an important signaling pathway between DNA damage repair and the cell metabolism, which contributed to the premature aging effects of PUVA, and may be generally applicable for a large category of chemotherapeutic reagents including many cancer drugs.

5-Deoxyribose is formed from 5′-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine (SAM) enzymes. The degradative fate of 5-deoxyribose is unknown. Here, we define a salvage pathway for 5-deoxyribose in bacteria, consisting of phosphorylation, isomerization, and aldol cleavage steps. Analysis of bacterial genomes uncovers widespread, unassigned three-gene clusters specifying a putative kinase, isomerase, and sugar phosphate aldolase. We show that the enzymes encoded by the Bacillus thuringiensis cluster, acting together in vitro, convert 5-deoxyribose successively to 5-deoxyribose 1-phosphate, 5-deoxyribulose 1-phosphate, and dihydroxyacetone phosphate plus acetaldehyde. Deleting the isomerase decreases the 5-deoxyribulose 1-phosphate pool size, and deleting either the isomerase or the aldolase increases susceptibility to 5-deoxyribose. The substrate preference of the aldolase is unique among family members, and the X-ray structure reveals an unusual manganese-dependent enzyme. This work defines a salvage pathway for 5-deoxyribose, a near-universal metabolite., 5-Deoxyribose is formed from 5′-deoxyadenosine, a toxic byproduct of radical S-adenosylmethionine enzymes. Here, the authors identify and biochemically characterize a bacterial salvage pathway for 5-deoxyribose, consisting of three enzymes, and solve the crystal structure of the key aldolase.

Summary Background Upregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined. Methods We explore human triple-negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received intravenous (i.v.) infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes. Findings TNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline, and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceed that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer. Conclusions Glucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the tricarboxylic acid (TCA) cycle locally within human breast tumors. Funding The clinical trial, genomics, and proteomics were funded by the Baylor Scott & White Dallas Foundation, Dallas, Texas. Metabolic analyses were supported by NIH grants 1DP1DK113643, R01CA163591, and P30CA072720.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects approximately seven million people, mainly in Latin America, and causes about 7000 deaths annually. The available treatments are unsatisfactory and search for more effective drugs against this pathogen is critical. In this context, the ribose 5-phosphate isomerase (Rpi) enzyme is a potential drug target mainly due to its function in the pentose phosphate pathway and its essentiality (previously shown in other trypanosomatids). In this study, we propose novel potential inhibitors for the Rpi of T. cruzi (TcRpi) based on a computer-aided approach, including structure-based and ligand-based pharmacophore modeling. Along with a substructural and similarity search, the selected pharmacophore hypotheses were used to screen the purchasable subset of the ZINC Database, yielding 20,183 candidate compounds. These compounds were submitted to molecular docking studies in the TcRpi and Human Rpi (HsRpi) active sites in order to identify potential selective inhibitors for the T. cruzi enzyme. After the molecular docking and ADME-T (absorption, distribution, metabolism, excretion and toxicity)/PAINS (pan-assay interference compounds) screenings, 211 molecules were selected as potential TcRpi inhibitors. Out of these, three compounds - ZINC36975961, ZINC63480117, and ZINC43763931 - were submitted to molecular dynamics simulations and two of them - ZINC36975961 and ZINC43763931- had good performance and made interactions with important active site residues over all the simulation time. These compounds could be considered potential TcRpi inhibitors candidates and also may be used as leads for developing new TcRpi inhibitors.

Packaging of DNA into the nucleosome core particle (NCP) is considered to exert constraints to all DNA-templated processes, including base excision repair where Pol β catalyzes two key enzymatic steps: 5′-dRP lyase gap trimming and template-directed DNA synthesis. Despite its biological significance, knowledge of Pol β activities on NCPs is still limited. Here, we show that removal of the 5′-dRP block by Pol β is unaffected by NCP constraints at all sites tested and is even enhanced near the DNA ends. In contrast, strong inhibition of DNA synthesis is observed. These results indicate 5′-dRP gap trimming proceeds unperturbed within the NCP; whereas, gap filling is strongly limited. In the absence of additional factors, base excision repair in NCPs will stall at the gap-filling step.

Phosphoribosylpyrophosphate synthetase (PRPPS) from the thermophilic bacterial strainThermus thermophilusHB27 catalyzes the synthesis of phosphoribosylpyrophosphate from ribose 5-phosphate and ATP, and belongs to the class I PRPPSs. The three-dimensional structure of the recombinant enzyme was solved at 2.2 Å resolution using crystals grown in microgravity from protein solution containing ATP, magnesium and sulfate ions. An ADP molecule was located in the active site of each subunit of the hexameric enzyme molecule and sulfate ions were located in both the active and allosteric sites. It was found that the catalytic loop that restricts the active-site area and is usually missing from the electron-density map of class I PRPPSs adopts different conformations in three independent subunits inT. thermophilusPRPPS. A closed conformation of the active site was found in one of subunits where the highly ordered catalytic β-hairpin delivers the Lys and Arg residues that are essential for activity directly to the ADP molecule, which occupies the ATP-binding site. A comparison of the conformations of the catalytic loop in the three independent subunits reveals a possible mode of transition from the open to the closed state of the active site during the course of the catalyzed reaction.

Background Transketolase-like 1 (TKTL1) plays an important role in pentose phosphate pathway (PPP) branch, the main pathway generating nicotinamide adenine dinucleotide phosphate (NADPH) and nucleotides for DNA synthesis. TKTL1 is closely related to DNA damage and has a close relationship with incidence and progression of cancers. Cisplatin is the main chemotherapeutic drug by inducing DNA damage. Whether TKTL1 knockdown additively complements cisplatin-induced cytotoxicity in nasopharyngeal carcinoma cells, however, remains largely undefined. Methods Lipofectamine 2000 was used to transfect si-TKTL1s with different sequences into the CNE2 and HONE1 cells. The mRNA and protein levels of TKTL1 were determined by qRT-PCR and western blot, respectively. MTT assay and flow cytometry were used to access the viability and apoptosis of CNE2 and HONE1 cells. The NADPH and ribose-5-phosphate levels in both CNE2 and HONE1 cells were determined by NADPH examination kit and HPCE analysis, respectively. The effect of TKTL1 knockdown and NADPH/ribose-5-phosphate supplement on DNA damage was assessed by using Comet assay. Results TKTL1 knockdown significantly decreased TKTL1 level in CNE2 and HONE1 cells. A significant decrease in cell viability and an obvious increase in cell apoptosis rate were found in si-TKTL1 + cisplatin group compared with si-TKTL1 group or si-control + cisplatin group. The levels of NADPH and ribose-5-phosphate in CNE1 and HONE1 cells were dramatically decreased in si-TKTL1 group compared with si-control group. TKTL1 knockdown additively complemented cisplatin-induced cytotoxicity, which was partly reversed by the supplements of NADPH and ribose-5-phosphate, including the increased survival rate, decreased apoptosis and DNA damage. Conclusions Knockdown of TKTL1 additively complements cisplatin-induced cytotoxicity in the nasopharyngeal carcinoma cells by inhibiting the levels of NADPH and ribose-5-phosphate, indicating that TKTL1 may be a promising target to improve the therapeutic effect combining with cisplatin for the patients with nasopharyngeal carcinoma.

Significance Many mutations that affect plastidial metabolism are embryo-lethal, as expected if the disrupted genes encode enzymes with essential housekeeping functions. However, some mutations that disrupt the plastidial oxidative pentose phosphate pathway (OPPP) cause developmental defects, as well as embryo arrest at the globular stage of development. We show that the OPPP provides the substrate for the pathway of purine synthesis, ribose-5-phosphate, and is thus essential for the generation of nucleic acids during the very early stages of embryo development. Inadequate purine synthesis leads to abnormal patterns of cell division in the embryo and blocks development beyond the globular stage. Therefore, defects in primary metabolic pathways can have profound consequences for development as well as simply reducing growth., Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)—required for purine nucleotide and histidine synthesis—and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.

D-ribose and D-arabinose differ only by the steric orientation of their C2-OH groups. The initial reactions and emergence of RNA depended on the position, reactivity, and flexibility of the C2-OH moiety in the ribose molecule. The steric relationship of the C2- and C3-OH groups favored the selection of ribose, ribonucleotide, and RNA synthesis and excluded the possibility of xenonucleic acid-based life on Earth. This brief review provides a hypothesis based on the absence of nucleotides and enzymes under prebiotic conditions and on the polymerization of ribose 5-phosphate units leading to the polarized formation of the ribose-phosphate backbone. The strong covalent bond formation in the sugar-phosphate backbone was followed by the somewhat less reactive interaction between ribose and nucleobase and supplemented by even weaker hydrogen-bonded and stacking interactions. This hypothesis proposes a scheme how prebiotic random-sequence RNA was formed under abiotic conditions and hydrolyzed to oligomers and nucleotides. The term random-sequence prebiotic RNA refers to nucleobases attached randomly to the ribose-phosphate backbone and not to cellular RNA sequences as proteins and cells did not probably exist at the time of abiotic RNA formation. It is hypothesized that RNA generated under abiotic conditions containing random nucleobases was hydrolyzed to nucleotides that served as a pool for the selected synthesis of genetic RNA.

Imidazole-4-acetic acid-ribotide (IAARP) is a putative neurotransmitter/modulator and an endogenous regulator of sympathetic drive, notably systemic blood pressure, through binding to imidazoline receptors. IAARP is present in neurons and processes throughout the CNS, but is particularly prevalent in regions that are involved in blood pressure control. The goal of this study was to determine whether IAARP is present in neurons in the caudal vestibular nuclei that participate in the vestibulo-sympathetic reflex (VSR) pathway. This pathway is important in modulating blood pressure upon changes in head position with regard to gravity, as occurs when humans rise from a supine position and when quadrupeds climb or rear. Sinusoidal galvanic vestibular stimulation was used to activate the VSR and cfos gene expression in VSR pathway neurons of rats. These subjects had previously received a unilateral FluoroGold tracer injection in the rostral or caudal ventrolateral medullary region. The tracer was transported retrogradely and filled vestibular neuronal somata with direct projections to the injected region. Brainstem sections through the caudal vestibular nuclei were immunostained to visualize FluoroGold, cFos protein, IAARP and glutamate immunofluorescence. The results demonstrate that IAARP is present in vestibular neurons of the VSR pathway, where it often co-localizes with intense glutamate immunofluorescence. The co-localization of IAARP and intense glutamate immunofluorescence in VSR neurons may represent an efficient chemoanatomical configuration, allowing the vestibular system to rapidly up- and down-modulate the activity of presympathetic neurons in the ventrolateral medulla, thereby altering blood pressure.

A phosphoribosyl anthranilate isomerase,TkTrpF, fromThermococcus kodakaraensiswas expressed inEscherichia coliand purified to homogeneity.TkTrpF was crystallized and its structure was determined by molecular replacement in two different space groups (C2 andP1) using data to 1.85 and 1.75 Å resolution, respectively.TkTrpF belongs to the class of TIM-barrel proteins. Structural comparison with other phosphoribosyl anthranilate isomerases (TrpFs) showed the highest structural similarity toPyrococcus furiosusTrpF. Similarly toP. furiosusTrpF,TkTrpF is a monomer in solution, in contrast to other thermophilic enzymes, which exist as functional dimers. Although in space groupP1TkTrpF crystallizes with two molecules in the asymmetric unit, the interface is highly improbable in solution. Potential factors for the thermostability ofTkTrpF were attributed to an increase in helical structure, an increased number of charged residues and an increase in the number of salt bridges.

Objective: to study immunophenotype and NAD- and NAD(P)-dependent dehydrogenase of blood lymphocytes activity indicators in children with hypertrophy of the pharyngeal tonsils (HPT).Methods: 57 children aged 1–3 years with HPT were examined. The focus group included 35 healthy children of the similar age. The number of СD3+-, СD4+-, СD8+-, СD16+/56+-, СD19+-cells in the blood was determined by flow cytometry. The activity of NAD(P)-dependent dehydrogenase was studied by the method of A. Savchenko and coauth. (1989).Results: The changes of immunophenotypic spectrum of peripheral blood lymphocytes in infants with HPT have been revealed. The increase of ribose-5-phosphate and NADN-dependent reactions of macromolecular synthesis, the reduction of malataspartat shunt role in cell energy, the reduction of anaerobic lactate dehydrogenase reaction, the compensatory increase in the glycerol-3-phosphate dehydrogenase activity, the high substrate flow of the citric acid cycle and the reduced level of glutathione have been fixed. The correlation analysis has showed increase in the number of correlations between indicators of investigated oxidoreductase activity in blood lymphocytes in children with HPT and the high level of correlation between the metabolic reactions of the mitochondrial compartment. Conclusion: the change of immunophenotype, enzymatic activity, correlation pattern of connection between intracellular enzymes of peripheral blood lymphocytes have been revealed in children aged 1–3 years with HPT.

De novo nucleotide biosynthesis is essential for maintaining cellular nucleotide pools, the suppression of which leads to genome instability. The metabolic enzyme transketolase (TKT) in the nonoxidative branch of the pentose phosphate pathway (PPP) regulates ribose 5-phosphate (R5P) levels and de novo nucleotide biosynthesis. TKT is required for maintaining cell proliferation in human liver cancer cell lines, yet the role of TKT in liver injury and cancer initiation remains to be elucidated. In this study, we generated a liver-specific TKT knockout mouse strain by crossing TKTflox/flox mice with albumin-Cre mice. Loss of TKT in hepatocytes protected the liver from diethylnitrosamine (DEN)-induced DNA damage without altering DEN metabolism. DEN treatment of TKT-null liver increased levels of R5P and promoted de novo nucleotide synthesis. More importantly, supplementation of dNTPs in primary hepatocytes alleviated DEN-induced DNA damage, cell death, inflammatory response, and cell proliferation. Furthermore, DEN and high-fat diet (HFD)–induced liver carcinogenesis was reduced in TKTflox/floxAlb-Cre mice compared with control littermates. Mechanistically, loss of TKT in the liver increased apoptosis, reduced cell proliferation, decreased TNFα, IL6, and STAT3 levels, and alleviated DEN/HFD-induced hepatic steatosis and fibrosis. Together, our data identify a key role for TKT in promoting genome instability during liver injury and tumor initiation. Significance: These findings identify transketolase as a novel metabolic target to maintain genome stability and reduce liver carcinogenesis.

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.

Methanol is a low-cost and abundantly available feedstock derived from natural gas and syngas. Although bioconversion holds promise for producing desired chemicals from methanol under economically viable operating conditions, the efficiency is limited by unfavorable kinetics of methanol oxidation and assimilation. Herein, artificial fusion proteins were engineered to enhance methanol bioconversion. Nicotinamide adenine dinucleotide (NAD)-dependent methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloisomerase (Phi) from different sources were first screened for catalytic activity. Next, we designed six fusion proteins using the best enzyme candidates and flexible linkers. Fusing Mdh with Hps or Hps-Phi increased the Vmax of methanol oxidation up to 5.8-fold, and enhanced methanol conversion to fructose-6-phosphate up to 1.3-fold. Interestingly, fusion engineering changed the polymerization states of proteins and produced larger multimers, which may be responsible for the changed catalytic characteristics. This fusion engineering approach can be coupled with other metabolic engineering strategies for enhanced methanol bioconversion to valuable chemicals.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM−1 · s−1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota. IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis. The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

Accumulation of nucleotide building blocks prior to and during S phase facilitates DNA duplication. Herein, we find that the anaphase-promoting complex/cyclosome (APC/C) synchronizes ribose-5-phosphate levels and DNA synthesis during the cell cycle. In late G1 and S phases, transketolase-like 1 (TKTL1) is overexpressed and forms stable TKTL1-transketolase heterodimers that accumulate ribose-5-phosphate. This accumulation occurs by asymmetric production of ribose-5-phosphate from the non-oxidative pentose phosphate pathway and prevention of ribose-5-phosphate removal by depleting transketolase homodimers. In the G2 and M phases after DNA synthesis, expression of the APC/C adaptor CDH1 allows APC/CCDH1 to degrade D-box-containing TKTL1, abrogating ribose-5-phosphate accumulation by TKTL1. TKTL1-overexpressing cancer cells exhibit elevated ribose-5-phosphate levels. The low CDH1 or high TKTL1-induced accumulation of ribose-5-phosphate facilitates nucleotide and DNA synthesis as well as cell cycle progression in a ribose-5-phosphate-saturable manner. Here we reveal that the cell cycle control machinery regulates DNA synthesis by mediating ribose-5-phosphate sufficiency., Ribose-5-phosphate (R5P) is required for DNA synthesis, but how this is regulated during cell cycle progression is unclear. Here the authors report that the cell cycle regulator APC/C-CDH1 synchronizes cell cycle progression with R5P-derived DNA synthesis by controlling TKTL1 stability

In the present study, we investigated the role of transketolase (TK) in the modulation of glycolaldehyde driven Maillard reactions. In vitro experiments with recombinant human TK reduced glycolaldehyde and glyoxal induced carbonyl stress and thereby suppressed the formation of advanced glycation endproducts up to 70% due to the enzyme-catalyzed conversion of glycolaldehyde to erythrulose. This was further substantiated by the use of 13C-labeled compounds. For the first time, glycolaldehyde and other sugars involved in the TK reaction were quantified in vivo and compared to nondiabetic uremic patients undergoing hemodialysis. Quantitation revealed amounts of glycolaldehyde up to 2 μM and highlighted its crucial role in the formation of AGEs in vivo. In this context, a LC-MS2 method for the comprehensive detection of sedoheptulose-7-phosphate, fructose-6-phosphate, ribose-5-phosphate, erythrose-4-phosphate, erythrulose, and glycolaldehyde in whole blood, plasma, and red blood cells was established and validated based on derivatization with 1-naphthylamine and sodium cyanoborohydride.

Aims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2−/− mice. Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130.

© 2018, The Author(s). 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is a class I aldolase that offers access to several building blocks for organic synthesis. It catalyzes the stereoselective C–C bond formation between acetaldehyde and numerous other aldehydes. However, the practical application of DERA as a biocatalyst is limited by its poor tolerance towards industrially relevant concentrations of aldehydes, in particular acetaldehyde. Therefore, the development of proper experimental conditions, including protein engineering and/or immobilization on appropriate supports, is required. The present review is aimed to provide a brief overview of DERA, its history, and progress made in understanding the functioning of the enzyme. Furthermore, the current understanding regarding aldehyde resistance of DERA and the various optimizations carried out to modify this property are discussed.

In this work, intimate contacts of riboses of mRNA stretch from nucleotides in positions +3 to +12 with respect to the first nucleotide of the P site codon were studied using cross-linking of short mRNA analogs with oxidized 3'-terminal riboses bound to human ribosomes in the complexes stabilized by codon-anticodon interactions and in the binary complexes. It was shown that in all types of complexes cross-links of the mRNA analogs to ribosomal protein (rp) uS3 occur and the yield of these cross-links does not depend on the presence of tRNA and on sequences of the mRNA analogs. Site of the mRNA analogs cross-linking in rp uS3 was mapped to the peptide in positions 55-64 that is located away from the mRNA binding site. Additionally, in complexes with P site-bound tRNA, riboses of mRNA nucleotides in positions +4 to +7 cross-linked to the C-terminal tail of rp uS19 displaying a contact specific to the decoding site of the mammalian ribosome, and tRNA bound at the A site completely blocked this cross-linking. Remarkably, rps uS3 and uS19 were also able to cross-link to the fragment of HCV IRES containing unstructured 3'-terminal part restricted by the AUGC tetraplet with oxidized 3'-terminal ribose. However, no cross-linking to rp uS3 was observed in the 48S preinitiation complex assembled in reticulocyte lysate with this HCV IRES derivative. The results obtained show an ability of rp uS3 to interact with single-stranded RNAs. Possible roles of rp uS3 region 55-64 in the functioning of ribosomes are discussed.

The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein.

While carbon-based nanomaterials such as graphene and carbon nanotubes (CNTs) have become popular in state-of-the-art nanotechnology, their biological safety and underlying molecular mechanism is still largely unknown. Experimental studies have been focused at the cellular level and revealed good correlations between cell's death and the application of CNTs or graphene. Using large-scale all-atom molecular dynamics simulations, we theoretically investigate the potential toxicity of graphene to a biological cell at molecular level. Simulation results show that the hydrophobic protein-protein interaction (or recognition) that is essential to biological functions can be interrupted by a graphene nanosheet. Due to the hydrophobic nature of graphene, it is energetically favorable for a graphene nanosheet to enter the hydrophobic interface of two contacting proteins, such as a dimer. The forced separation of two functional proteins can disrupt the cell's metabolism and even lead to the cell's mortality.

d -Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d -ribulose-5-phosphate and d -arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d -arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d -glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani , contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d -arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d -arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium. IMPORTANCE The genome of Clostridium tetani , a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d -arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d -arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d -glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani , an organism that does not produce lipopolysaccharide.

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi) RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV-and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 10(10) EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p

SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

The 2-deoxy- d -ribose-5-phosphate aldolase (DERA) is a synthetically attractive enzyme because of its ability to perform C C-couplings stereoselectively, the enzyme uses acetaldehyde as nucleophile and thus produces true aldols rather than ketols, and may add two acetaldehyde molecules onto one electrophile. However, DERA produces crotonaldehyde as side reaction from acetaldehyde which is then an irreversible inhibitor forming a covalent Michael -adduct within the active site in particular with cysteine 47 (Dick et al., 2016). This inhibition can be resolved by mutating C47 to non-nucleophile amino acids. Still, the inhibition is not an on-off-feature and the present mutagenesis study illustrates that there must be a C47-independent inactivation mechanism. As a practical result: The virtually fully resistant mutant C47L was found, which shows no loss in stereoselectivity, − this renders this variant as promising catalyst.