Measuring Laccase Activity and Melanin Production in Cryptococcus neoformans.

Melanin is a complex dark pigment synthetized by the phenoloxidase enzyme laccase in Cryptococcus neoformans. In vitro, this enzyme oxidizes exogenous catecholamines to produce melanin that may be secreted or incorporated into the fungal cell wall. This pigment has multiple roles in C. neoformans virulence during its interaction with different hosts and probably also in protecting fungal cells in the environment against predation and oxidative and radiation stresses, among others. However, it is important to note that laccase also has melanin-independent roles in C. neoformans interactions with host cells. In this chapter, we describe a quantitative laccase assay and a method for evaluating the kinetics of melanin production in C. neoformans colonies.

Molecular modelling studies and in vitro enzymatic assays identified A 4-(nitrobenzyl)guanidine derivative as inhibitor of SARS-CoV-2 Mpro.

Scientists and researchers have been searching for drugs targeting the main protease (Mpro) of SARS-CoV-2, which is crucial for virus replication. This study employed a virtual screening based on molecular docking to identify benzoylguanidines from an in-house chemical library that can inhibit Mpro on the active site and three allosteric sites. Molecular docking was performed on the LaSMMed Chemical Library using 88 benzoylguanidine compounds. Based on their RMSD values and conserved pose, three potential inhibitors (BZG1, BZG2, and BZG3) were selected. These results indicate that BZG1 and BZG3 may bind to the active site, while BZG2 may bind to allosteric sites. Molecular dynamics data suggest that BZG2 selectively targets allosteric site 3. In vitro tests were performed to measure the proteolytic activity of rMpro. The tests showed that BZG2 has uncompetitive inhibitory activity, with an IC50 value of 77 µM. These findings suggest that benzoylguanidines possess potential as Mpro inhibitors and pave the way towards combating SARS-Cov-2 effectively.

Quantification of GTPase Cycling Rates of GTPases and GTPase:Effector Mixtures Using GTPase Glo Assays.

In different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay-the GTPase Glo assay by Promega-for investigation of GTPase-effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration-dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conducting GTPase Glo assays Support Protocol 1: Analyzing GTPase assays to correlate luminescence with remaining GTP Support Protocol 2: Fitting GTPase assay data to obtain GTPase cycling rates.

Preparation, assay, and application of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 for ß-hydroxyl amino acid derivatives.

ß-Hydroxy-α-amino acids (ßHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cß positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse ßHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of ßHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable ßHAAs. Among these naturally occurring transformations, L-threonine transaldolases present potential biocatalysts to generate ßHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehyde to give 4-fluorothreonine and acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various ßHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of ßHAAs from various aldehydes and L-Thr using FTaseMA.

Microwell fluoride assay screening for enzymatic defluorination.

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F-. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 µL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

Application of Fenton chemistry in electrochemical determination of pyrophosphatase activity and fluoride.

Fenton chemistry has aroused widespread concern due to its application in the green oxidation and mineralization of organic wastes. Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate ions (PPi) and provides a thermodynamic driving force for many biosynthetic reactions. Fluoride (F-) is widely applied to fight against tooth decay and reduce cavities. The electrochemical determination of PPase activity and F- was realized based on Fenton chemistry in this work. Glassy carbon electrode modified with poly (azure A) and acetylene black (GCE/PAA-AB) was fabricated. Hydroxyl radicals (∙OH) that were generated from a Cu2+-catalyzed Fenton-type reaction could oxidize PAA in the near-neutral medium, leading to a great increase of the cathodic peak current (Ipc). A coordination reaction between PPi and Cu2+ exerted a negative effect on Fenton reaction and hindered the Ipc enhancement. Cu2+-PPi complex was decomposed due to the hydrolysis of PPi induced by PPase, which caused the reappearance of the notably increased current response. F- could effectively inhibit PPase activity. As a result, the stable Cu2+-PPi complex remained and the high Ipc suffered from the decline again. The Ipc difference was used for the highly sensitive determination of PPase activity in the content range of 0.001-20 mU mL-1 with a detection of limit (LOD) at 0.6 µU mL-1 and that of F- in the concentration range of 0.01-100 µM with a LOD at 7 nM. The proposed PPase and F- sensor displayed a good selectivity, stability and reproducibility, and a high accuracy.

Fluorescent metal nanoclusters: From luminescence mechanism to applications in enzyme activity assays.

Metal nanoclusters (MNCs) have outstanding fluorescence property and biocompatibility, which show widespread applications in biological analysis. Particularly, evaluation of enzyme activity with the fluorescent MNCs has been developed rapidly within the past several years. In this review, we first introduced the fluorescent mechanism of mono- and bi-metallic nanoclusters, respectively, whose interesting luminescence properties are mainly resulted from electron transfer between the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) energy levels. Meanwhile, the charge migration within the structure occurs through ligand-metal charge transfer (LMCT) or ligand-metal-metal charge transfer (LMMCT). On such foundation, diverse enzyme activities were rigorously evaluated, including three transferases and nine hydrolases, in turn harvesting rapid research progresses within past 5 years. Finally, we summarized the design strategies for evaluating enzyme activity with the MNCs, presented the major issues and challenges remained in the relevant research, coupled by showing some improvement measures. This review will attract researchers dedicated to the studies of the MNCs and provide some constructive insights for their further applications in enzyme analysis.

An Enzyme Assay Kit for GABA Quantification in Plant Tissues.

Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200 mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues.

Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Detection of terminal deoxynucleotidyl transferase activity based on self-mediated nucleic acid elongation and elemental labeling inductively coupled plasma-mass spectrometry.

Terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase, is recognized as a promising biomarker for acute leukemia. Herein, taking the advantage of the self-mediated strand elongation property of TdT, a simple and sensitive method for TdT activity assay was developed based on gold nanoparticles (AuNPs) labeling inductively coupled plasma mass spectrometry (ICP-MS). In the presence of TdT, the primer DNA on magnetic beads is elongated with an adenine-rich single stranded long chain that can label poly-thymine modified AuNPs. After acid elution, the labeled AuNPs were detected by ICP-MS, and the signal intensity of 197Au reflected the TdT activity. Under the optimal conditions, the limit of detection for TdT activity is down to 0.054 U mL-1, along with good selectivity and strong tolerance to other interfering proteins. Furthermore, it achieves a straightforward and accurate detection of TdT activity in acute lymphoblastic leukemia cells without sample pre-processing and tool enzyme addition. Therefore, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia therapeutics.

Biogenic Polymer-Based Fluorescent Assemblies: Versatile Platforms for Ultrasensitive ATP Detection and Enzyme Assay.

Here, we investigated the optical properties of biocompatible supramolecular assemblies formed through electrostatic interactions between anionic fluorescent dyes and biogenic polymers. The dynamic equilibrium between the monomeric form (fluorescent) and aggregates (nonfluorescent) of dye molecules is responsible for the stimuli-responsive behavior of these polymer composites, which can respond to changes in pH, temperature, and ionic strength. Furthermore, we employed supramolecular assemblies for the purpose of turn-on fluorescence sensing of adenosine triphosphate (ATP) at physiological pH. Notably, no interference was observed even in the presence of well-known competing analytes such as pyrophosphate. In addition to its outstanding selectivity, the present system can detect ATP at concentrations as low as 4.8 nM. The superior detection capabilities are achieved through multiple interactions with biogenic polymers, involving the adenine ring, ribose unit (through hydrogen bonding), and phosphate groups (via charge pairing) of ATP. Given the remarkable sensitivity to ATP, we have applied the present system for the detection of a dephosphorylating enzyme, alkaline phosphatase.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Preparation of ER Microsomes from Arabidopsis thaliana.

Microsomes are vesicles derived from the endoplasmic reticulum (ER) when cells are broken down in the lab. These microsomes are a valuable tool to study a variety of ER functions such as protein and lipid synthesis in vitro.Here we describe a protocol to isolate ER-derived microsomes Arabidopsis thaliana seedlings and exemplify the use of these purified microsomes in enzyme assays with the auxin precursors tryptophan (Trp) or indole-3-pyruvic acid (IPyA) to quantify auxin synthetic capacity in microsomal and cytosolic fractions.

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

17ß-Hydroxysteroid dehydrogenases types 1 and 2: Enzymatic assays based on radiometric and mass-spectrometric detection.

The 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) has a key role in estrogen biosynthesis as it catalyzes the reduction of estrone to the most potent estrogen, estradiol. Estradiol has a high affinity for estrogen receptors and thus stimulates their transactivation, which leads to cell proliferation and numerous other effects. HSD17B2 catalyzes the oxidation of estradiol to the less potent estrone, thereby decreasing estrogen receptor activation, which results in reduction of estrogen-associated effects. HSD17B1 and HSD17B2 overexpressing E.coli homogenates or recombinant enzymes can be used for screening and development of drugs against various pathologies such as cancer, endometriosis or osteoporosis. Here we describe the preparation of HSD17B1 and HSD17B2 bacterial homogenates and purified recombinant HSD17B1 protein as enzyme sources as well as enzymatic assays based on radiometric and mass-spectrometric detection for enzyme characterization.

Aldo keto-reductase family 1C members 1 through 4 recombinant enzyme purification and enzyme assay.

Aldo-keto reductase family 1C (AKR1C) members transform steroids via their 3-, 17-, and 20-ketosteroid reductase activities. The biochemical study of these enzymes can help to inform their roles in hormone-dependent diseases and develop therapeutic inhibitors. This work describes a protocol to purify AKR1C1-4 members from a bacterial expression system using two chromatography steps. It also describes the basis of discontinuous assays to measure steroid conversion.

Sensitive Silver-Enhanced Microplate Apta-Enzyme Assay of Sb3+ Ions in Drinking and Natural Waters.

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3',5,5'-tetramethylbenzidine substrate, generating an optical signal. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag+ at the stage of A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ was determined to be 8-135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb3+ in the range of 109.0-126.2% and 99.6-106.1%, respectively.