Substrate Preference Research Articles

Analysis and Prediction of Chymotrypsin Substrate Preferences through Large Data Acquisition with Target-Free mRNA Display.

Oral delivery of peptide therapeutics is limited by degradation by gut proteases like chymotrypsin. Existing databases of peptidases are limited in size and do not enable systematic analyses of protease substrate preferences, especially for non-natural amino acids. Thus, stability optimization of hit compounds is time and resource intensive. To accelerate the stability optimization of peptide ligands, we generated large datasets of chymotrypsin-resistant peptides via mRNA display to create a predictive model for chymotrypsin-resistant sequences. Through analysis of enriched motifs, we recapitulate known chymotrypsin cleavage sites, reveal positionally dependent effects of monomers on peptide cleavage, and report previously unidentified protective and destabilizing residues. We then developed a machine-learning-based modelpredicting peptide resistance to chymotrypsin cleavage and validated both model performance and the NGS experimental data by measuring chymotrypsin half-lives for a subset of peptides. Finally, we simulated stability predictions on non-natural amino acids through a leucine hold-out model and observed robust performance. Overall, we demonstrate the utility of mRNA display as a tool for big data generation and show that pairing mRNA display with machine learning yields valuable predictions for chymotrypsin cleavage. Expansion of this workflow to additional proteases could provide complementary predictive models that focus future peptide drug discovery efforts.

Carbon preference by Cupriavidus necator for growth and accumulation phases: Heterotrophic vs. autotrophic metabolisms

Cupriavidus necator is a mixotrophic microbe capable of metabolizing both organic carbon (heterotrophic) and CO2 (autotrophic) for cell growth and biodegradable plastic synthesis. This study investigates C. necator's behavior under mixotrophic conditions for cell growth and polyhydroxyalkanoate (PHA) accumulation. Various concentrations of organic substrates (acetate and butyrate from 10 to 1000 mg/L) and gaseous substrates (H2, O2, CO2) were examined to monitor C. necator's substrate preferences. The results indicated a clear preference for organic carbon over CO2 during cell growth. At relatively low organic concentrations (10–100 mg/L), gas utilization began after complete consumption of organic substrates. On the other hand, higher concentrations (500–1000 mg/L) led to a shift from the heterotrophic to mixotrophic metabolism around the 10-h mark due to the abundance of substrates. During PHA accumulation, C. necator initially favored the gaseous substrates (H2 and CO2) before shifting to the organic substrates, with mixotrophic behavior observed immediately at low organic concentrations. Additionally, high CO2 partial pressure (>0.4 atm) inhibited microbial growth in both autotrophic and heterotrophic metabolisms. These findings highlight C. necator's adaptability under mixotrophic conditions and demonstrate the potential for mass-scale applications, such as microbial electrolysis cells to optimize biopolymer production and substrate utilization.

Comparison Between Simple Batch and Fed-Batch Bioreactor Cultivation of Recombinant BCG

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process.

Lipoxygenase activity in tropical fish: Changes during surimi processing and some biochemical characterization in lizardfish (Saurida tumbil).

The lipoxygenase (LOX) activity of major tropical fish used for surimi production, including threadfin bream (TB), lizardfish (LZ), and goatfish (GF), was measured in the gills, skin, and muscle. The highest LOX activity was observed in the LZ samples (p<0.05), with the gills exhibiting the greatest activity at 376.56U/mg (p<0.05). The highest peroxide value was detected in the TB samples, particularly in the gills (p<0.05). Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were the main polyunsaturated fatty acids in all the tissues and surimi. The total lipid and DHA contents of washed mince reduced considerably after the screw press process. Although LOX activity decreased during surimi production, a residual activity of 21.33U/g was observed in the finished surimi. LOX was partially purified and characterized from LZ gills. The purification was conducted using two successive chromatographic steps, Sephacryl S-200 and diethylaminoethyl (DEAE)-sepharose, resulting in a 3.52% yield and a 22.43-fold increase in purity. The optimum activity was found at 25°C and pH 7.5, with pH stability between 6.0 and 8.5. The relatively high thermal stability at 4°C-10°C suggested that LOX might contribute to fish lipid oxidation during cold storage. The enzyme was thermally inactivated at 60°C. The preferred substrate was EPA. LOX from the LZ gills was inhibited by 1mM ethylenediaminetetraacetic acid and activated by 1mM Fe2+, Na+, and Ca2+. PRACTICAL APPLICATION: Elucidating lipoxygenase activity and lipid oxidation in various tropical fish tissues, as well as understanding the characteristics of LOX, can help take appropriate postharvest actions to afford high-quality surimi.

Adaptation in the Anthropocene: How behavioural choice and colour change enables chameleon prawns to camouflage on non-native seaweeds.

Animals combine colour change and behavioural choices to enhance concealment and adapt to changes in habitat in time and space. However, non-native and invasive habitat-forming plants and seaweeds can change the landscape, challenging animals to remain camouflaged, especially when the colour of the new habitat differs from the native backgrounds.The chameleon prawn (Hippolyte varians) exhibits remarkable colour variation and effective camouflage against different native seaweeds in shallow tidepools. Individuals optimize crypsis by choosing colour-matching seaweeds and changing colour over time. In some locations, the prawn's native habitats are now replaced by non-native seaweeds of different coloration and structure, making it important to understand whether substrate choice and colour change facilitates the occupation of these seaweeds and enables prawns to camouflage against new backgrounds.Using image analysis and visual modelling of a fish predator, we assessed the colour variation and camouflage of chameleon prawns occupying the non-native seaweeds brown wireweed (Sargassum muticum) and pink harpoon weed (Asparagopsis armata) in southwest UK. We performed laboratory trials to examine whether prawns maintain their preference for colour-matching native substrates when given a choice between them and non-native seaweeds, and if they can change their coloration to improve camouflage against non-native substrates.Prawns exhibit phenotypic diversity and camouflage that varied with the non-native seaweed species, with low colour variation and effective camouflage on pink harpoon weed, but high colour diversity and reduced concealment against brown wireweed. Prawns choose non-native seaweeds when the alternative native substrate provides mismatching coloration, but they did not exhibit any preference between colour-matching native and non-native seaweeds. Once in non-native habitats, prawns change their appearance over a few days to match the background, sometimes faster than when changing on native seaweeds of contrasting coloration.Although human activities considerably modify the coloration of marine landscapes by increasing the establishment of non-native seaweeds, the impacts of those changes appear less severe for colour-changing species, such as chameleon prawns, and will depend on the similarity between the colour of the new substrates and the original native backgrounds, and how animals use behaviour and physiology to match new habitats.

Genetic Variation in Trophic Avoidance Behaviour Shows Fruit Flies are Generally Attracted to Bacterial Substrates.

Pathogen avoidance behaviours are often assumed to be an adaptive host defence. However, there is limited experimental data on heritable, intrapopulation phenotypic variation for avoidance, a strong prerequisite for adaptive responses to selection. We investigated trophic pathogen avoidance in 122 inbred Drosophila melanogaster lines, and in a derived outbred population. Using the FlyPAD system, we tracked the feeding choice that flies made between substrates that were either clean or contained a bacterial pathogen. We uncovered significant, but weakly heritable variation in the preference index amongst fly lines. However, instead of avoidance, most lines demonstrated a preference for substrates containing several bacterial pathogens, showing avoidance only for extremely high bacterial concentrations. Bacterial preference was not associated with susceptibility to infection and was retained in flies with disrupted immune signalling. Phenotype-genotype association analysis indicated several novel genes (CG2321, CG2006, and ptp99A) associated with increased preference for the bacterial substrate, while the amino-acid transporter sobremesa was associated with greater aversion. Given the known fitness benefits of consuming high-protein diets, our results suggest that bacterial attraction may instead reflect a dietary preference for protein over carbohydrate. More work quantifying intrapopulation variation in avoidance behaviours is needed to fully assess its importance in host-pathogen evolutionary ecology.

New Toolset of Reporters Reveals That Glycogen Granules Are Neutral Substrates of Bulk Autophagy in Komagataella phaffii.

Glycogen, a branched polysaccharide organized into glycogen granules (GGs), is delivered from the cytoplasm to the lysosomes of hepatocytes by STBD1-driven selective autophagy (glycophagy). Recently, we developed Komagataella phaffii yeast as a simple model of GG autophagy and found that it proceeds non-selectively under nitrogen starvation conditions. However, another group, using Saccharomyces cerevisiae as a model, found that glycogen is a non-preferred cargo of nitrogen starvation-induced bulk autophagy. To clarify cargo characteristics of K. phaffii GGs, we used the same glycogen synthase-based reporter (Gsy1-GFP) of GG autophagy in K. phaffii as was used in S. cerevisiae. The K. phaffii Gsy1-GFP marked the GGs and reported on their autophagic degradation during nitrogen starvation, as expected. However, unlike in S. cerevisiae, glycogen synthase-marked GGs were delivered to the vacuole and degraded there with the same efficiency as a cytosolic glycogen synthase in glycogen-deficient cells, suggesting that glycogen is a neutral cargo of bulk autophagy in K. phaffii. We verified our findings with a new set of reporters based on the glycogen-binding CBM20 domain of human STBD1. The GFP-CBM20 and mCherry-CBM20 fusion proteins tagged GGs, reported about the autophagy of GGs, and confirmed that GGs in K. phaffii are neither preferred nor non-preferred substrates of bulk autophagy. They are its neutral substrates.

Illuminating Substrate Preferences of Promiscuous F420H2-Dependent Dehydroamino Acid Reductases with 4-Track mRNA Display.

Stereoselective reduction of dehydroamino acids is a common biosynthetic strategy to introduce d-amino acids into peptidic natural products. The reduction, often observed during the biosynthesis of lanthipeptides, is performed by dedicated dehydroamino acid reductases (dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide, flavin, or F420H2 coenzymes as hydride donors, and little is known about the catalysis performed by the latter family proteins. Here, we perform a bioinformatics-guided identification and large-scale in vitro characterization of five F420H2-dependent dhAARs. We construct an mRNA display-based pipeline for ultrahigh throughput substrate specificity profiling of the enzymes. The pipeline relies on a 4-track selection strategy to deliver large quantities of clean data, which were leveraged to build accurate substrate fitness models. Our results identify a remarkably promiscuous enzyme, referred to as MaeJC, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition requirements. We integrate MaeJC into a thiopeptide biosynthetic pathway to produce d-amino acids-containing thiopeptides, demonstrating the utility of MaeJC for the programmable installation of d-amino acids in ribosomal peptides.

Rare in Ukraine and new for the Ukrainian Carpathians species of macrofungi recorded in the Kamenystyi protected area (Hutsulshchyna National Nature Park)

A list of 20 rare in Ukraine species of macrofungi (Ascomycota and Basidiomycota) recorded in 2012–2023 in the Kamenisty protected area (Hutsulshchyna National Nature Park, Ivano-Frankivsk Region) is provided. Of them, 12 records are new for the Ukrainian Carpathians. Information about habitats, substrate preferences, ecological features and general distribution of the fungi is provided. Eight species (Clitocybe angustissima, Cortinarius praestans, C. prasinus, Discina fastigiata, Hygrophorus poetarum, Lactarius acerrimus, Leucopaxillus gentianeus and Tricholoma pardinum), which are red-listed in a number of European countries, are recommended to be included in the Red List of fungi of Ivano-Frankivsk Region.

Two ubiquitous aldo-keto reductases in the genus Papaver support a patchwork model for morphine pathway evolution

The evolution of morphinan alkaloid biosynthesis in plants of the genus Papaver includes permutation of several processes including gene duplication, fusion, neofunctionalization, and deletion resulting in the present chemotaxonomy. A critical gene fusion event resulting in the key bifunctional enzyme reticuline epimerase (REPI), which catalyzes the stereochemical inversion of (S)-reticuline, was suggested to precede neofunctionalization of downstream enzymes leading to morphine biosynthesis in opium poppy (Papaver somniferum). The ancestrally related aldo-keto reductases 1,2-dehydroreticuline reductase (DRR), which occurs in some species as a component of REPI, and codeinone reductase (COR) catalyze the second and penultimate steps, respectively, in the pathway converting (S)-reticuline to morphine. Orthologs for each enzyme isolated from the transcriptomes of 12 Papaver species were shown to catalyze their respective reactions in species that capture states of the metabolic pathway prior to key evolutionary events, including the gene fusion event leading to REPI, thus suggesting a patchwork model for pathway evolution. Analysis of the structure and substrate preferences of DRR orthologs in comparison with COR orthologs revealed structure-function relationships underpinning the functional latency of DRR and COR orthologs in the genus Papaver, thus providing insights into the molecular events leading to the evolution of the pathway.

Proteomics-based computational modelling of myocardial metabolism for personalized phenotyping and outcome prediction in advanced heart failure

Abstract Background Perturbations of myocardial metabolism and energy depletion are well-established hallmarks of heart failure (HF), yet methods for their systematic assessment remain limited in humans [1]. Computational modelling of the heart of individual patients represents an emerging precision medicine approach for personalized diagnosis of specific disease mechanisms and tailored clinical decision-making [2,3]. Purpose To determine the ability of computational modelling of patient-specific myocardial metabolism to assess individual bioenergetic phenotypes and their prognostic implications in HF. Methods Based on proteomics-derived enzyme intensity profiles in cardiac biopsies, we recently developed a computational model of myocardial metabolism capturing all pathways along energy-delivering substrates are processed to generate ATP [4]. Using this framework, we studied the individual energetic state in 17 sex-matched nonfailing controls and 48 patients with advanced HF (LVEF 20±10%, 72.9% non-ischemic aetiology, 52.1% females, 29.8% diabetics) undergoing heart transplantation (n=25) or ventricular assist device (VAD) implantation (n=23). The bioenergetic impact of alterations in substrate availability and myocardial workload were simulated, and the model’s ability to predict myocardial reverse remodelling after VAD implantation was assessed. Results Compared to controls, HF patients had a reduced ATP production capacity (p&amp;lt;0.01), although there was considerable interindividual variance, ranging from severely decreased to normal values. While overall oxygen consumption was lower in failing hearts, the amount of ATP generated per mole of oxygen did not differ (p=0.16). Maximum uptake capacity of fatty acids, glucose, and amino acids was comparable. By contrast, maximum uptake capacity of ketone bodies and lactate was reduced in the failing heart (p for both &amp;lt;0.05). There was a shift from fatty acid to glucose utilization in most HF patients, depending on substrate availability and myocardial workload. The ratio of fatty acid to glucose oxidation distinguished patients with LVEF recovery &amp;gt;10% after VAD implantation (C-index 0.93, p&amp;lt;0.01; Picture 1B) and correlated significantly with improvement in post-operative LVEF (r=0.67, p&amp;lt;0.01). ATP production capacity was not associated with reverse remodelling after VAD implantation. Conclusions Computational modelling identified a subset of advanced HF patients with preserved myocardial metabolism despite a similar degree of systolic dysfunction. Myocardial substrate preference but not ATP production capacity was associated with myocardial recovery after VAD implantation, which suggests a potential for substrate manipulation as a therapeutic approach in those with deranged fuel use. Computational assessment of myocardial metabolism in HF may improve understanding of disease heterogeneity, individual risk stratification, and guidance of personalized clinical decision-making in the future.

Progress in Protein-Based Hydrogels for Flexible Sensors: Insights from Casein.

In recent years, the rapid advancement of flexible sensors as the cornerstone of flexible electronics has propelled a flourishing evolution within the realm of flexible electronics. Unlike traditional flexible devices, hydrogel flexible sensors have characteristic advantages such as biocompatibility, adhesion, and adjustable mechanical properties and have similar properties to human skin. Especially, biobased hydrogels have become the preferred substrate material for flexible sensors due to increased environmental pressures caused by the scarcity of petrochemical resources. In this regard, proteins possess advantages such as diverse amino acid compositions, adjustable advanced structures, chemical modifiability, the application of protein engineering techniques, and the ability to respond to various external stimuli. These enable the hydrogels constructed from them to have greater designability, flexibility, and adaptability. As a result, their applications in manufacturing various types of sensors have experienced rapid growth. This work systematically reviews the sensing mechanism of protein-based hydrogels, focusing on the preparation of protein-based hydrogels and the optimization of flexible sensors mainly from the perspective of a typical type of animal-derived protein casein. In addition, while the potential of casein is recognized, the limitations of casein-based hydrogels in flexible sensor applications are explored, and insights are provided into the development trends of next-generation sensors based on casein-based hydrogel materials.

Biochemical Characterization of an Arabinoside Monophosphate Specific 5′-Nucleotidase-like Enzyme from Streptomyces antibioticus

To investigate the function of the gene penF in the pentostatin and vidarabine (Ara-A) biosynthetic gene cluster in Streptomyces antibioticus NRRL 3238, PenF was recombinantly expressed and characterized. Enzymatic characterization of the enzyme demonstrated that PenF exhibited metal-dependent nucleoside 5ʹ-monophosphatase activity, showing a substrate preference for arabinose nucleoside 5ʹ-monophosphate over 2ʹ-deoxyribonucleoside 5ʹ-monophosphate and ribonucleoside 5ʹ-monophosphate. Metal ions such as Mg2+ and Mn2+ significantly enhanced enzyme activity, whereas Zn2+, Cu2+, and Ca2+ inhibited it. For vidarabine 5′-monophosphate, the Km and kcat values were determined to be 71.5 μM and 33.9 min−1, respectively. The kcat/Km value was 474.1 mM−1·min−1 for vidarabine 5-monophosphate and was 68-fold higher than that for 2′-deoxyadenosine 5′-monophosphate. Comparative sequence alignment and structural studies suggested that residues outside the primary substrate-binding site are responsible for this substrate specificity. In conclusion, PenF’s activity toward vidarabine 5ʹ-monophosphate likely plays a role in the dephosphorylation of precursors during Ara-A biosynthesis.

Two-Dimensional Covalent Organic Frameworks with Carbazole-Embedded Frameworks Facilitate Photocatalytic and Electrocatalytic Processes.

Catalytic technologies are pivotal in enhancing energy efficiency, promoting clean energy production, and reducing energy consumption in the chemical industry. The pursuit of novel catalysts for renewable energy is a long-term goal for researchers. In this work, we synthesized three two-dimensional covalent organic frameworks (COFs) featuring electron-rich carbazole-based architectures and evaluated their catalytic performance in photocatalytic organic reactions and electrocatalytic oxygen reduction reactions (ORRs). Pyrene-functionalized COF, termed as FCTD-TAPy, demonstrated excellent photocatalytic performance for amino oxidation coupling and showed a remarkable preference for substrates with electron-withdrawing groups (up to >99% Conv. and >99% Sel). Furthermore, FCTD-TAPy favored a four-electron transfer pathway during the ORR and exhibited favorable reaction kinetics (51.07 mV/dec) and a high turnover frequency (0.011 s-1). In contrast, the ORR of benzothiadiazole-based FCTD-TABT favored a two-electron transfer pathway, which exhibited a maximum double-layer capacitance of 14.26 mF cm-2, a Tafel slope of 53.01 mV/dec, and a hydrogen peroxide generation rate of 70.3 mmol g-1 h-1. This work underscores the potential of carbazole-based COFs as advanced catalytic materials and offers new insights into the design of metal-free COFs for enhanced catalytic performance.

Expanding the biosynthesis spectrum of hydroxy fatty acids: unleashing the potential of novel bacterial fatty acid hydratases

BackgroundHydroxy fatty acids represent an emerging class of compounds with promising applications in the chemical, medicinal and functional food sectors. The challenges associated with their chemical synthesis have spurred exploration of biological synthesis as an alternative route, particularly through the use of fatty acid hydratases. Fatty acid hydratases catalyse the regioselective addition of a hydrogen atom and a hydroxyl group from a water molecule to the carbon–carbon cis-double bond of unsaturated fatty acids to form hydroxy fatty acids. Despite having been discovered in the early 1960s, previous research has primarily focused on characterizing single fatty acid hydratase variants with a limited range of substrates. Comprehensive studies that systematically examine and compare the characteristics of multiple variants of fatty acid hydratases are still lacking.ResultsIn this study, we employed an integrated bioinformatics workflow to identify 23 fatty acid hydratases and characterized their activities against nine unsaturated fatty acid substrates using whole-cell biotransformation assays. Additionally, we tested a dual-protein system involving two fatty acid hydratases of distinct regioselectivity and demonstrated its suitability in enhancing the biosynthesis of di-hydroxy fatty acids.ConclusionsOur study demonstrates that fatty acid hydratases can be classified into three subtypes based on their regioselectivity and provides insights into their preferred substrate structures. These understandings pave ways for the design of optimal fatty acid hydratase variants and bioprocesses for the cost-efficient biosynthesis of hydroxy fatty acids.

CG17192 is a Phospholipase That Regulates Signaling Lipids in the Drosophila Gut upon Infection.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly (Drosophila melanogaster). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

An easy and sensitive assay for acetohydroxyacid synthases based on the simultaneous detection of substrates and products in a single step.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes the first step in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine, pathways being present in plants and microorganisms, but not in animals. Thus, AHAS is an important target for numerous herbicides and, more recently, for the development of antimicrobial agents. The need to develop new and optimized herbicides and pharmaceuticals requires a detailed understanding of the biochemistry of AHAS. AHAS transfers an activated two-carbon moiety derived from pyruvate to either pyruvate or 2-oxobutyrate as acceptor substrates, forming 2-acetolactate or 2-acetohydroxy-2-butyrate, respectively. Various methods have been described in the literature to biochemically characterize AHAS with respect to substrate preferences, substrate specificity, or kinetic parameters. However, the simultaneous detection and quantification of substrates and unstable products of the AHAS-catalyzed reaction have always been a challenge. Using AHAS isoform II from Escherichia coli, we have developed a sensitive assay for AHAS-catalyzed reactions that uses derivatization with ethyl chloroformate to stabilize and volatilize all reactants in the aqueous solution and detect them by gas chromatography coupled to flame ionization detection or mass spectrometry. This assay allows us to characterize the product formation in reactions in single and dual substrate reactions and the substrate specificity of AHAS, and to reinterpret previous biochemical observations. This assay is not limited to the AHAS-catalyzed reactions, but should be applicable to studies of many metabolic pathways.

Sequence diversity in the monooxygenases involved in oxime production in plant defense and signaling: a conservative revision in the nomenclature of the highly complex CYP79 family.

Cytochrome P450 monooxygenases of the CYP79 family catalyze conversion of specific amino acids into oximes feeding into a variety of metabolic plant pathways. Here we present an extensive phylogenetic tree of the CYP79 family built on carefully curated sequences collected across the entire plant kingdom. Based on a monophyletic origin of the P450s, a set of evolutionarily distinct branches was identified. Founded on the functionally characterized CYP79 sequences, sequence features of the individual substrate recognition sites (SRSs) were analyzed. Co-evolving amino acid residues were identified using co-evolutionary sequence analysis. SRS4 possesses a specific sequence pattern when tyrosine is a substrate. Except for the CYP79Cs and CYP79Fs, substrate preferences toward specific amino acids could not be assigned to specific subfamilies. The highly diversified CYP79 tree, reflecting recurrent independent evolution of CYP79s, may relate to the different roles of oximes in different plant species. The sequence differences across individual CYP79 subfamilies may facilitate the in vivo orchestration of channeled metabolic pathways based on altered surface charge domains of the CYP79 protein. Alternatively, they may serve to optimize dynamic interactions with oxime metabolizing enzymes to enable optimal ecological interactions. The outlined detailed curation of the CYP79 sequences used for building the phylogenetic tree made it appropriate to make a conservative phylogenetic tree-based revision of the naming of the sequences within this highly complex cytochrome P450 family. The same approach may be used in other complex P450 subfamilies. The detailed phylogeny of the CYP79 family will enable further exploration of the evolution of function in these enzymes.

When are you measuring soil β‐glucosidase activities in cropping systems?

AbstractIn situ soil respiration is driven by annual patterns of temperature and soil moisture, but what about extracellular enzyme activities responsible for depolymerizing soil organic matter? We conducted biweekly measurements of potential soil β‐glucosidase activities during a 4‐month period from March soil thawing through July in annually cropped field plots in eastern South Dakota. Our objective was to determine the best sampling time to resolve the effects of crop rotational diversity on soil microbial activities. Potential β‐glucosidase activities were elevated immediately following soil thaw, peaked in May, and declined to their lowest value in mid‐summer. Temperature and precipitation had no value in predicting enzyme activities; however, enzyme activities were affected by crop rotational diversity and responded to current crop and previous crop. These findings are pertinent to the use of soil extracellular enzymes in soil health assessments and as indicators of microbial substrate preference with implications for soil carbon stabilization.

Physicochemical and thermodynamic properties of purified rhodanese from A. welwitschiae LOT1 and the cyanide detoxification potential of the enzyme.

Rhodanese, the primary cyanide-detoxifying enzyme, plays a crucial role in mitigating the harmful effects of cyanide present in various industrial waste materials, such as battery manufacturing effluents. The bioremediation of cyanide-contaminated environments relies on efficient detoxification mechanisms, making rhodanese a valuable enzyme for biotechnological applications. This research aimed to investigate the biochemical properties of purified rhodanese produced by Aspergillus welwitschiae LOT1, a fungal strain with promising cyanide detoxification capabilities. The purified rhodanese was obtained through fermentation, precipitation, and chromatographic separations, resulting in a homogeneous band of approximately 58kDa with a specific activity of 374 RU/mg, 28-fold purification, and 14% recovery. The enzyme exhibited optimal cyanide detoxification at pH 7 and 60°C, with stability observed between 30 and 50°C and pH 8-10. All metal ions examined except for Cu2+ enhanced the cyanide-degrading ability of rhodanese. Notably, the enzyme demonstrated a high substrate preference for Na2S2O3 and followed a first-order kinetic model and free energy, ΔG of 61.3kJ/mol, making it a promising candidate for biotechnological applications. Overall, this study provides valuable insights into the biochemical properties of rhodanese from A. welwitschiae LOT1, highlighting its potential for efficient cyanide detoxification and bioremediation.