Structural Basis of Hydrogenotrophic Methanogenesis.

Most methanogenic archaea use the rudimentary hydrogenotrophic pathway-from CO2 and H2 to methane-as the terminal step of microbial biomass degradation in anoxic habitats. The barely exergonic process that just conserves sufficient energy for a modest lifestyle involves chemically challenging reactions catalyzed by complex enzyme machineries with unique metal-containing cofactors. The basic strategy of the methanogenic energy metabolism is to covalently bind C1 species to the C1 carriers methanofuran, tetrahydromethanopterin, and coenzyme M at different oxidation states. The four reduction reactions from CO2 to methane involve one molybdopterin-based two-electron reduction, two coenzyme F420-based hydride transfers, and one coenzyme F430-based radical process. For energy conservation, one ion-gradient-forming methyl transfer reaction is sufficient, albeit supported by a sophisticated energy-coupling process termed flavin-based electron bifurcation for driving the endergonic CO2 reduction and fixation. Here, we review the knowledge about the structure-based catalytic mechanism of each enzyme of hydrogenotrophic methanogenesis.

Protein cost minimization promotes the emergence of coenzyme redundancy.

SignificanceMetabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes [e.g., NAD(H) and NADP(H)] associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling and structural and sequence analysis indicates that coenzyme redundancy may not be essential for metabolism but could rather constitute an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems.

The energetics and evolution of oxidoreductases in deep time.

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation-reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox (https://protein-redox-potential.web.app), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation-reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.

Angiotensin converting enzyme inhibitor and coenzyme Q10 as adjunctive treatment for patients with ventricular septal rupture following late onset myocardial infarction: a case report.

Ventricular Septal Rupture (VSR) is a rare complication of acute myocardial infarction and has a high mortality rate. Surgery is the definitive treatment. However, in hospitals with limited facilities, treating acute myocardial infarction patients with ventricular septal rupture, is challenging. A 74-year-old woman came to the emergency room of Dr. Koesma General Hospital, Tuban, East Java in December, 2019 with late-onset Acute Myocardial Infarction. On the following day, a new holosystolic murmur was heard in the left lower sternal border with palpable thrill. Transthoracic echocardiography showed VSR with severe pulmonary hypertension. This was followed by a drop in the blood pressure to 80/50 mmHg. The blood pressure was dependent on vasopressors until lisinopril and coenzyme Q10 were introduced. After 3 months, the haemodynamics of the patient were stable. This proved that the use of angiotensin-converting enzyme and coenzyme Q10 promotes more energy production, enables tissue healing and leads to balanced remodelling to increase the survival rate in cases of non-surgical treatment.

B vitamin supply in plants and humans: the importance of vitamer homeostasis.

B vitamins are a group of water-soluble micronutrients that are required in all life forms. With the lack of biosynthetic pathways, humans depend on dietary uptake of these compounds, either directly or indirectly, from plant sources. B vitamins are frequently given little consideration beyond their role as enzyme accessory factors and are assumed not to limit metabolism. However, it should be recognized that each individual B vitamin is a family of compounds (vitamers), the regulation of which has dedicated pathways. Moreover, it is becoming increasingly evident that individual family members have physiological relevance and should not be sidelined. Here, we elaborate on the known forms of vitamins B1 , B6 and B9 , their distinct functions and importance to metabolism, in both human and plant health, and highlight the relevance of vitamer homeostasis. Research on B vitamin metabolism over the past several years indicates that not only the total level of vitamins but also the oft-neglected homeostasis of the various vitamers of each B vitamin is essential to human and plant health. We briefly discuss the potential of plant biology studies in supporting human health regarding these B vitamins as essential micronutrients. Based on the findings of the past few years we conclude that research should focus on the significance of vitamer homeostasis - at the organ, tissue and subcellular levels - which could improve the health of not only humans but also plants, benefiting from cross-disciplinary approaches and novel technologies.

Effect of femtosecond laser interaction with human fibroblasts: a preliminary study.

In in vitro methods and cell culture models, femtosecond (fs) laser interaction has been employed to assess its effect on the proliferation and morphology of human skin fibroblasts. We cultured a primary human skin fibroblast cell line on a glass plate, passages 17-23. The cells were irradiated with a 90-fs laser at a wavelength of 800 nm and a repetition rate of 82 MHz. The target received an average power of 320 mW for 5, 20, and 100 s, corresponding to the radiation exposures of 22.6, 90.6, and 452.9 J/cm2, respectively. Using a laser scanning microscopy technique, the photon densities were measured to be 6.4 × 1018, 2.6 × 1019, and 1.3 × 1020 photons/cm2 in a spot area of 0.07 cm2; the recorded spectra were obtained from the laser interaction after 0.00, 1.00, 25.00, and 45.00 h. The cell count and morphological changes showed that the cultured cells were affected by laser irradiation under photon stress; some fibroblasts were killed, while others were injured and survived. We discovered evidence of the formation of several coenzyme compounds, such as flavin (500-600 nm), lipopigments (600-750 nm), and porphyrin (500-700 nm). This study is motivated by the future development of a novel, ultra-short fs laser system and the need to develop a basic in vitro understanding of photon-human cell interaction. The cell proliferation indicated that cells are partly killed or wounded. The exposure of fibroblasts to fs laser fluence up to 450 J/cm2 accelerates cell growth of the viable residual cell.

Role of coenzymes in cancer metabolism.

Cancer is a heterogeneous set of diseases characterized by the rewiring of cellular signaling and the reprogramming of metabolic pathways to sustain growth and proliferation. In past decades, studies were focused primarily on the genetic complexity of cancer. Recently, increasing number of studies have discovered several mutations among metabolic enzymes in different tumor cells. Most of the enzymes are regulated by coenzymes, organic cofactors, that function as intermediate carrier of electrons or functional groups that are transferred during the reaction. However, the precise role of cofactors is not well elucidated. In this review, we discuss several metabolic enzymes associated to cancer metabolism rewiring, whose inhibition may represent a therapeutic target. Such enzymes, upon expression or inhibition, may impact also the coenzymes levels, but only in few cases, it was possible to direct correlate coenzymes changes with a specific enzyme. In addition, we also summarize an up-to-date information on biological role of some coenzymes, preclinical and clinical studies, that have been carried out in various cancers and their outputs.

The key role of the NAD biosynthetic enzyme nicotinamide mononucleotide adenylyltransferase in regulating cell functions.

The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes a reaction central to all known NAD biosynthetic routes. In mammals, three isoforms with distinct molecular and catalytic properties, different subcellular and tissue distribution have been characterized. Each isoform is essential for cell survival, with a critical role in modulating NAD levels in a compartment-specific manner. Each isoform supplies NAD to specific NAD-dependent enzymes, thus regulating their activity with impact on several biological processes, including DNA repair, proteostasis, cell differentiation, and neuronal maintenance. The nuclear NMNAT1 and the cytoplasmic NMNAT2 are also emerging as relevant targets in specific types of cancers and NMNAT2 has a key role in the activation of antineoplastic compounds. This review recapitulates the biochemical properties of the three isoforms and focuses on recent advances on their protective function, involvement in human diseases and role as druggable targets.

Unveiling the mechanisms and biosynthesis of a novel nickel-pincer enzyme.

The nickel-pincer nucleotide (NPN) coenzyme, a substituted pyridinium mononucleotide that tri-coordinates nickel, was first identified covalently attached to a lysine residue in the LarA protein of lactate racemase. Starting from nicotinic acid adenine dinucleotide, LarB carboxylates C5 of the pyridinium ring and hydrolyzes the phosphoanhydride, LarE converts the C3 and C5 carboxylates to thiocarboxylates, and LarC incorporates nickel to form a C-Ni and two S-Ni bonds, during the biosynthesis of this cofactor. LarB uses a novel carboxylation mechanism involving the transient formation of a cysteinyl-pyridinium adduct. Depending on the source of the enzyme, LarEs either catalyze a sacrificial sulfur transfer from a cysteinyl side chain resulting in the formation of dehydroalanine or they utilize a [4Fe-4S] cluster bound by three cysteine residues to accept and transfer a non-core sulfide atom. LarC is a CTP-dependent enzyme that cytidinylylates its substrate, adds nickel, then hydrolyzes the product to release NPN and CMP. Homologs of the four lar genes are widely distributed in microorganisms, with some species containing multiple copies of larA whereas others lack this gene, consistent with the cofactor serving other functions. Several LarA-like proteins were shown to catalyze racemase or epimerase activities using 2-hydroxyacid substrates other than lactic acid. Thus, lactate racemase is the founding member of a large family of NPN-containing enzymes.

On the Evolutionary History of the Twenty Encoded Amino Acids.

α-Amino acids are essential molecular constituents of life, twenty of which are privileged because they are encoded by the ribosomal machinery. The question remains open as to why this number and why this 20 in particular, an almost philosophical question that cannot be conclusively resolved. They are closely related to the evolution of the genetic code and whether nucleic acids, amino acids, and peptides appeared simultaneously and were available under prebiotic conditions when the first self-sufficient complex molecular system emerged on Earth. This report focuses on prebiotic and metabolic aspects of amino acids and proteins starting with meteorites, followed by their formation, including peptides, under plausible prebiotic conditions, and the major biosynthetic pathways in the various kingdoms of life. Coenzymes play a key role in the present analysis in that amino acid metabolism is linked to glycolysis and different variants of the tricarboxylic acid cycle (TCA, rTCA, and the incomplete horseshoe version) as well as the biosynthesis of the most important coenzymes. Thus, the report opens additional perspectives and facets on the molecular evolution of primary metabolism.

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications.

Pterins are an inseparable part of living organisms. Pterins participate in metabolic reactions mostly as tetrahydropterins. Dihydropterins are usually intermediates of these reactions, whereas oxidized pterins can be biomarkers of diseases. In this review, we analyze the available data on the quantum chemistry of unconjugated pterins as well as their photonics. This gives a comprehensive overview about the electronic structure of pterins and offers some benefits for biomedicine applications: (1) one can affect the enzymatic reactions of aromatic amino acid hydroxylases, NO synthases, and alkylglycerol monooxygenase through UV irradiation of H4pterins since UV provokes electron donor reactions of H4pterins; (2) the emission properties of H2pterins and oxidized pterins can be used in fluorescence diagnostics; (3) two-photon absorption (TPA) should be used in such pterin-related infrared therapy because single-photon absorption in the UV range is inefficient and scatters in vivo; (4) one can affect pathogen organisms through TPA excitation of H4pterin cofactors, such as the molybdenum cofactor, leading to its detachment from proteins and subsequent oxidation; (5) metal nanostructures can be used for the UV-vis, fluorescence, and Raman spectroscopy detection of pterin biomarkers. Therefore, we investigated both the biochemistry and physical chemistry of pterins and suggested some potential prospects for pterin-related biomedicine.

Cofactors are Remnants of Life's Origin and Early Evolution.

The RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite system of DNA, RNA, and proteins was preceded by one consisting solely of RNA, which both stored genetic information and performed the molecular functions encoded by that genetic information. Current research into a potential RNA World revolves around the catalytic properties of RNA-based enzymes, or ribozymes. Well before the discovery of ribozymes, Harold White proposed that evidence for a precursor RNA world could be found within modern proteins in the form of coenzymes, the majority of which contain nucleobases or nucleoside moieties, such as Coenzyme A and S-adenosyl methionine, or are themselves nucleotides, such as ATP and NADH (a dinucleotide). These coenzymes, White suggested, had been the catalytic active sites of ancient ribozymes, which transitioned to their current forms after the surrounding ribozyme scaffolds had been replaced by protein apoenzymes during the evolution of translation. Since its proposal four decades ago, this groundbreaking hypothesis has garnered support from several different research disciplines and motivated similar hypotheses about other classes of cofactors, most notably iron-sulfur cluster cofactors as remnants of the geochemical setting of the origin of life. Evidence from prebiotic geochemistry, ribozyme biochemistry, and evolutionary biology, increasingly supports these hypotheses. Certain coenzymes and cofactors may bridge modern biology with the past and can thus provide insights into the elusive and poorly-recorded period of the origin and early evolution of life.

Coenzymes and Their Role in the Evolution of Life.

The evolution of coenzymes, or their impact on the origin of life, is fundamental for understanding our own existence. Having established reasonable hypotheses about the emergence of prebiotic chemical building blocks, which were probably created under palaeogeochemical conditions, and surmising that these smaller compounds must have become integrated to afford complex macromolecules such as RNA, the question of coenzyme origin and its relation to the evolution of functional biochemistry should gain new impetus. Many coenzymes have a simple chemical structure and are often nucleotide-derived, which suggests that they may have coexisted with the emergence of RNA and may have played a pivotal role in early metabolism. Based on current theories of prebiotic evolution, which attempt to explain the emergence of privileged organic building blocks, this Review discusses plausible hypotheses on the prebiotic formation of key elements within selected extant coenzymes. In combination with prebiotic RNA, coenzymes may have dramatically broadened early protometabolic networks and the catalytic scope of RNA during the evolution of life.

[Determination of the fluorescence intensity of coenzymes NADH and FAD in the skeletal muscle of the rat depending on the post-mortem interval]. / Opredelenie intensivnosti fluorestsentsii kofermentov NADN i FAD v skeletnoi myshtse krysy v zavisimosti ot davnosti nastupleniia smerti.

Aim of the study is to identify patterns of variations of the fluorescence intensity of NADH (reduced nicotinamide adenine dinucleotide) and FAD (oxidized flavin adenine dinucleotide) in the skeletal muscle depending on the time since death. For the evaluation of fluorescence intensity of the studied coenzymes, laser-induced spectroscopy in situ was used. We revealed the dynamic of the fluorescence intensity of NADH and FAD in the skeletal muscle of a rat at different times during the post-mortem period, and theoretically justified the observed phenomena. The results obtained allow us to consider the studied indicators as a potential criterion for determining the post-mortem interval.

The tissue profile of metabolically active coenzyme forms of vitamin B12 differs in vitamin B12-depleted rats treated with hydroxo-B12 or cyano-B12.

Recent rat studies show different tissue distributions of vitamin B12 (B12), administered orally as hydroxo-B12 (HO-B12) (predominant in food) and cyano-B12 (CN-B12) (common in supplements). Here we examine male Wistar rats kept on a low-B12 diet for 4 weeks followed by a 2-week period on diets with HO-B12 (n 9) or CN-B12 (n 9), or maintained on a low-B12 diet (n 9). Plasma B12 was analysed before, during and after the study. The content of B12 and its variants (HO-B12, glutathionyl-B12, CN-B12, 5'-deoxyadenosyl-B12 (ADO-B12), and methyl-B12 (CH3-B12)) were assessed in the tissues at the end of the study. A period of 4 weeks on the low-B12 diet reduced plasma B12 by 58 % (from median 1323 (range 602-1791) to 562 (range 267-865) pmol/l, n 27). After 2 weeks on a high-B12 diet (week 6 v. week 4), plasma B12 increased by 68 % (HO-B12) and 131 % (CN-B12). Total B12 in the tissues accumulated differently: HO-B12>CN-B12 (liver, spleen), HO-B12

Methanogenesis on Early Stages of Life: Ancient but Not Primordial.

Of the six known autotrophic pathways, the Wood-Ljungdahl pathway (WL) is the only one present in both the acetate producing Bacteria (homoacetogens) and the methane producing Archaea (hydrogenotrophic methanogens), and it has been suggested that WL is one of the oldest metabolic pathways. However, only the so-called carbonyl branch is shared by Archaea and Bacteria, while the methyl branch is different, both in the number of reactions and enzymes, which are not homologous among them. In this work we show that some parts of the methyl branch of archaeal Wood-Ljungdahl pathway (MBWL) are present in bacteria as well as in non-methanogen archaea, although the tangled evolutionary history of MBWL cannot be traced back to the Last Common Ancestor. We have also analyzed the different variants of methanogenesis (hydrogenotrophic, acetoclastic and methylotrophic pathways), and concluded that each of these pathways, and every different enzyme or subunit (in the case of multimeric enzymes), has their own intricate evolutionary history. Our study supports the scenario of hydrogenotrophic methanogenesis being older than the other variants, albeit not old enough to be present in the last archaeal common ancestor.

Review on Microreactors for Photo-Electrocatalysis Artificial Photosynthesis Regeneration of Coenzymes.

In recent years, with the outbreak of the global energy crisis, renewable solar energy has become a focal point of research. However, the utilization efficiency of natural photosynthesis (NPS) is only about 1%. Inspired by NPS, artificial photosynthesis (APS) was developed and utilized in applications such as the regeneration of coenzymes. APS for coenzyme regeneration can overcome the problem of high energy consumption in comparison to electrocatalytic methods. Microreactors represent a promising technology. Compared with the conventional system, it has the advantages of a large specific surface area, the fast diffusion of small molecules, and high efficiency. Introducing microreactors can lead to more efficient, economical, and environmentally friendly coenzyme regeneration in artificial photosynthesis. This review begins with a brief introduction of APS and microreactors, and then summarizes research on traditional electrocatalytic coenzyme regeneration, as well as photocatalytic and photo-electrocatalysis coenzyme regeneration by APS, all based on microreactors, and compares them with the corresponding conventional system. Finally, it looks forward to the promising prospects of this technology.

Oligomerization and aggregation of NAP-22 with several metal ions.

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

Coenzymes and the primary and specialized metabolism interface.

In plants, primary and specialized metabolism have classically been distinguished as either essential for growth or required for survival in a particular environment. Coenzymes (organic cofactors) are essential for growth but their importance to specialized metabolism is often not considered. In line with the recent proposal of viewing primary and specialized metabolism as an integrated whole rather than segregated lots with a defined interface, we highlight here the importance of collating information on the regulation of coenzyme supply with metabolic demands using examples of vitamin B derived coenzymes. We emphasize that coenzymes can have enormous influence on the outcome of metabolic as well as engineered pathways and should be taken into account in the era of synthetic biology.

A new limit for blood metabolite analysis using 1H NMR spectroscopy.

Human blood is the most widely used biospecimen in the clinic and the metabolomics field. While both mass spectrometry and NMR spectroscopy are the two premier analytical platforms in the metabolomics field, NMR exhibits several unsurpassed characteristics for blood metabolite analysis, the most important of which are its ability to identify unknown metabolites and its quantitative nature. However, the relatively small number of metabolites accessible by NMR has restricted the scope of its applications. Enhancing the limit of identified metabolites in blood will therefore greatly impact NMR-based metabolomics. Continuing our efforts to address this major issue, our current study describes the identification of 12 metabolites, which expands the number of quantifiable blood metabolites by ~15%. These results, in combination with our earlier efforts, now provide access to nearly 90 metabolites, which is the highest to date for a simple 1D 1H NMR experiment that is widely used in the metabolomics field. Metabolites were identified based on the comprehensive investigation of human blood and plasma using 1D/2D NMR techniques. The newly identified metabolites were validated based on chemical shift databases, spectra of authentic compounds obtained under conditions identical to blood/plasma, and, finally, spiking experiments using authentic compounds. Considering the high reproducibility of NMR and the sensitivity of chemical shifts to altered sample conditions, experimental protocols and peak annotations are provided for the newly identified metabolites, which serve as a template for identification of blood metabolites for routine applications. Separately, the identified metabolites were evaluated for their sensitivity to preanalytical conditions. The results reveal that among the newly identified metabolites, inosine monophosphate (IMP) and nicotinamide are associated with labile coenzymes and their levels are sensitive to preanalytical conditions. The study demonstrates the expansion of quantifiable blood metabolites using NMR to a new height and is expected to greatly impact blood metabolomics.