Enzyme-like catalysis by single chain nanoparticles that use transition metal cofactors

We report a modular approach in which a noncovalently cross-linked single chain nanoparticle (SCNP) selectively binds catalyst “cofactors” and substrates to increase both the catalytic activity of a Cu-catalyzed alkyne-azide...

Designer Compartment for Artificial Metalloenzymes through Controlled Liquid-Liquid Phase Separation

Artificial metalloenzymes with different protein scaffolds, cofactors and functions have been prepared to expand the natural enzymatic repertoire with abiotic reactions. However, due to the sensitivity of metal centers toward various biomolecules, especially glutathione, low activity and turnover of artificial metalloenzymes in vivo are systematic problems not fully solved. Apart from straightforward routes such as the use of neutralizing agents, metal cofactors modification and directed evolution, one may notice that nature can create isolated microenvironments for diverse biological processes within cells. Following this way, here we report the in vivo assembly of artificial metalloenzymes based on HaloTag-SNAPTag fusion protein. These metalloenzymes have metal cofactors bound on protein interfaces, and can trigger liquid-liquid phase separation to form liquid condensates inside Escherichia coli. These condensates serve as membraneless, isolated compartments for artificial metalloenzymes to efficiently perform intracellular catalysis, mediating abiotic unmasking, coupling and polymerization reactions. The cellular compartmentalization also enables spatial control of reactions, either facilitating a cascade reaction within the confined spaces, or concurrent reactions with spatial separation. Such engineered Escherichia coli can work as whole-cell catalyst with confined metal species, colonizing at mice intestine to effect in vivo abiotic transformations with a lower chance of heavy metal poisoning. These results represent a systematic strategy to stabilize and potentiate artificial metalloenzyme in vivo, with potential applications in fields such as non-natural metabolism, fermentation and drug delivery.

Superoxide Dismutase: A Key Enzyme for the Survival of Intracellular Pathogens in Host

Superoxide dismutase (SOD) is a crucial enzyme required to maintain the redox potential of the cells. It plays a vital role in protecting normal cells from reactive oxygen species (ROS) produced during many intracellular pathogens infections. SOD removes excess superoxide radicals (O2−) by converting them to hydrogen peroxide (H2O2) and molecular oxygen (O2). Several superoxide dismutase enzymes have been identified based on the metal ion as a cofactor. Human SOD differs from the intracellular pathogens in having Cu/Zn and Mn as metal cofactors. However, SOD of intracellular pathogens such as Trypanosoma, Leishmania, Plasmodium, and Mycobacterium have iron (Fe) as metal cofactors. Iron Superoxide Dismutase (FeSOD) is an essential enzyme in these pathogens that neutralizes the free radical of oxygen (O−) and prevents the formation of Peroxynitrite anion (ONOO−), helping the pathogens escape from redox-based cytotoxic killing. Moreover, most intracellular bacteria hold MnSOD or FeSOD in their cytoplasm such as Salmonella and Staphylococcus, whereas periplasm of some pathogenic bacteria and fungi are also cofactors with Cu/Zn and identified as CuZnSOD. This chapter will review the various types SOD present in intracellular pathogens and their role in the survival of these pathogens inside their host niche.

Gene Fitness of Azotobacter vinelandii Under Diazotrophic Growth

Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly amenable to genetic manipulation, helping to unravel the intricate importance of different proteins involved in the process of BNF, including the biosynthesis of cofactors that are essential to assembling the complex metal cofactors that catalyze the difficult reaction of nitrogen fixation. Additionally, A. vinelandii accomplishes this feat while growing as an obligate aerobe, differentiating it from many of the nitrogen-fixing bacteria that are associated with plant roots. The ability to function in the presence of oxygen makes A. vinelandii suitable for application in various potential biotechnological schemes. In this study, we employed transposon sequencing (Tn-seq) to measure the fitness defects associated with disruptions of various genes under nitrogen-fixing dependent growth, versus growth with extraneously provided urea as a nitrogen source. The results allowed us to probe the importance of more than 3800 genes, revealing that many genes previously believed to be important, can be successfully disrupted without impacting cellular fitness. Importance These results provide insights into the functional redundancy in A. vinelandii , while also providing a direct measure of fitness for specific genes associated with the process of BNF. These results will serve as a valuable reference tool in future studies to uncover the mechanisms that govern this process.

Direct Observation of Electrically Conductive Pili Emanating from Geobacter sulfurreducens

Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport.

Molecular Details on Multiple Cofactor Containing Redox Metalloproteins Revealed by Infrared and Resonance Raman Spectroscopies

Vibrational spectroscopy and in particular, resonance Raman (RR) spectroscopy, can provide molecular details on metalloproteins containing multiple cofactors, which are often challenging for other spectroscopies. Due to distinct spectroscopic fingerprints, RR spectroscopy has a unique capacity to monitor simultaneously and independently different metal cofactors that can have particular roles in metalloproteins. These include e.g., (i) different types of hemes, for instance hemes c, a and a3 in caa3-type oxygen reductases, (ii) distinct spin populations, such as electron transfer (ET) low-spin (LS) and catalytic high-spin (HS) hemes in nitrite reductases, (iii) different types of Fe-S clusters, such as 3Fe-4S and 4Fe-4S centers in di-cluster ferredoxins, and (iv) bi-metallic center and ET Fe-S clusters in hydrogenases. IR spectroscopy can provide unmatched molecular details on specific enzymes like hydrogenases that possess catalytic centers coordinated by CO and CN− ligands, which exhibit spectrally well separated IR bands. This article reviews the work on metalloproteins for which vibrational spectroscopy has ensured advances in understanding structural and mechanistic properties, including multiple heme-containing proteins, such as nitrite reductases that house a notable total of 28 hemes in a functional unit, respiratory chain complexes, and hydrogenases that carry out the most fundamental functions in cells.

Nitrogen Fixation and Diazotrophs – A Review

Nitrogen fixation involves formation of ammonium from N2, which needs a high input of energy. Biological nitrogen fixation utilizes the enzyme nitrogenase and ATP to fix nitrogen. Nitrogenase contains a Fe-protein and a Mo-Fe-protein and other metal cofactors. Soil diazotrophs possess the function of fixing atmospheric N2 into biologically available ammonium in ecosystems. In Aechaea, nitrogen fixation has been reported in some methanogens such as Methanobacteriales, Methanococcales, and Methanosarcinales. Community structure and diversity of diazotrophic are correlated with soil pH. All known organisms which involve in nitrogen-fixing which are called diazatrophs are prokaryotes, and both bacterial and archaeal domains are responsible for that. Diazotrophs are categorized into two main groups namely: root-nodule bacteria and plant growth-promoting rhizobacteria. Diazotrophs include free living bacteria, such as Azospirillum, Cupriavidus, and some sulfate reducing bacteria, and symbiotic diazotrophs such Rhizobium and Frankia. Two important parameters which may affect diazotroph communities are temperature and soil moisture in different seasons. To have sustainable agriculture, replacing expensive chemical nitrogen fertilizers with environmentally friendly ways is the most accepted practice.

Fe-S cofactors in the SARS-CoV-2 RNA-dependent RNA polymerase are potential antiviral targets

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease 2019 (COVID-19), uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.

Photorespiration: The Futile Cycle?

Photorespiration, or C2 photosynthesis, is generally considered a futile cycle that potentially decreases photosynthetic carbon fixation by more than 25%. Nonetheless, many essential processes, such as nitrogen assimilation, C1 metabolism, and sulfur assimilation, depend on photorespiration. Most studies of photosynthetic and photorespiratory reactions are conducted with magnesium as the sole metal cofactor despite many of the enzymes involved in these reactions readily associating with manganese. Indeed, when manganese is present, the energy efficiency of these reactions may improve. This review summarizes some commonly used methods to quantify photorespiration, outlines the influence of metal cofactors on photorespiratory enzymes, and discusses why photorespiration may not be as wasteful as previously believed.

Nitrogen fixation in a diazotrophic post-bloom situation in the Baltic Sea

<p>The Baltic Sea is characterised as a semi-enclosed brackish Sea that has experienced increased eutrophication, hypoxia, and increased temperature over the last ~100 years making Baltic Sea one of the most severely impacted oceanic environment by climate change. Biological fixation of dinitrogen gas (N<sub>2</sub>) is an essential process to make atmospheric N<sub>2</sub> available for marine life. This process is carried out by specialised organisms called diazotrophs and is catalysed by the energetic-consuming enzyme nitrogenase. Nitrogenases exist in three subtypes depending on their metal cofactors, (1) the most common molybdenum-dependent (Nif), (2) the vanadium-dependent (Vnf) and (3) the Iron-Iron-dependent nitrogenase (Anf). To date, the effect of climate change on those three enzyme subtypes and their potential role a future ocean is yet to be explored. The predicted ongoing oxygen loss in the ocean may limit Mo's availability and trigger a shift from the abundant Nif-type nitrogenase to Vnf or Anf and, therefore, a potential shift in the diazotrophic community. This study explored the climate change-related pressures on N<sub>2</sub> fixation and the diazotrophic community based on nifH and vnf/anfD amplicons. At the time of sampling, we found a post-bloom high-nutrient low-chlorophyll situation. Cyanobacterial groups, Nodularia and UCYN-A, dominated the diazotrophic community and showed a horizontal where UCYN-A were the dominant fixers at 20 m. Based on alternative nitrogenases amplicons, Rhodopseudomonas was the dominating microbe in the surface water. This paper presents the first hint of active nitrogenases in surface water and further establish UCYN-A as a significant player in Baltic Sea primary production.</p>