Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes.

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01.

Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.

Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.

Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.

Expression, purification, kinetics, and crystallization of non-heme mononuclear iron enzymes: Biphenyl, Phthalate, and Terephthalate dioxygenases.

Non-heme iron oxygenases constitute a versatile enzyme family that is crucial for incorporating molecular oxygen into diverse biomolecules. Despite their importance, only a limited number of these enzymes have been structurally and functionally characterized. Surprisingly, there remains a significant gap in understanding how these enzymes utilize a typical architecture and reaction mechanism to catalyze a wide range of reactions. Improving our understanding of these catalysts holds promise for advancing both fundamental enzymology and practical applications. This chapter aims to outline methods for heterologous expression, enzyme preparation, in vitro enzyme assays, and crystallization of biphenyl dioxygenase, phthalate dioxygenase and terephthalate dioxygenase. These enzymes catalyze the dihydroxylation of biphenyl, phthalate and terephthalate molecules, serving as a model for functional and structural analysis of other non-heme iron oxygenases.

Biochemical and biophysical approaches to characterization of the aromatic amino acid hydroxylases.

The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase utilize a non-heme iron to catalyze the hydroxylation of the aromatic rings of their amino acid substrates, with a tetrahydropterin serving as the source of the electrons necessary for the monooxygenation reaction. These enzymes have been subjected to a variety of biochemical and biophysical approaches, resulting in a detailed understanding of their structures and mechanism. We summarize here the experimental approaches that have led to this understanding.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis coupled with High Performance Liquid Chromatography (HPLC) for subsequent detection. This method can be performed in anaerobic glove bag settings. It requires readily available HPLC instrumentation for ligand quantitation and is adaptable to meet the needs of a variety of substrate affinity measurements.

Functional analysis of an α-ketoglutarate-dependent non-heme iron oxygenase in fungal meroterpenoid biosynthesis.

α-Ketoglutarate-dependent non-heme iron (α-KG NHI) oxygenases compose one of the largest superfamilies of tailoring enzymes that play key roles in structural and functional diversifications. During the biosynthesis of meroterpenoids, α-KG NHI oxygenases catalyze diverse types of chemical reactions, including hydroxylation, desaturation, epoxidation, endoperoxidation, ring-cleavage, and skeletal rearrangements. Due to their catalytic versatility, keen attention has been focused on functional analyses of α-KG NHI oxygenases. This chapter provides detailed methodologies for the functional analysis of the fungal α-KG NHI oxygenase SptF, which plays an important role in the structural diversification of andiconin-derived meroterpenoids. The procedures included describe how to prepare the meroterpenoid substrate using a heterologous fungal host, measure the in vitro enzymatic activity of SptF, and how to perform structural and mutagenesis studies on SptF. These protocols are also applicable to functional analyses of other α-KG NHI oxygenases.

Spectroscopic and computational studies of a bifunctional iron- and 2-oxoglutarate dependent enzyme, AsqJ.

Iron and 2-oxoglutarate dependent (Fe/2OG) enzymes exhibit an exceedingly broad reaction repertoire. The most prevalent reactivity is hydroxylation, but many other reactivities have also been discovered in recent years, including halogenation, desaturation, epoxidation, endoperoxidation, epimerization, and cyclization. To fully explore the reaction mechanisms that support such a diverse reactivities in Fe/2OG enzyme, it is necessary to utilize a multi-faceted research methodology, consisting of molecular probe design and synthesis, in vitro enzyme assay development, enzyme kinetics, spectroscopy, protein crystallography, and theoretical calculations. By using such a multi-faceted research approach, we have explored reaction mechanisms of desaturation and epoxidation catalyzed by a bi-functional Fe/2OG enzyme, AsqJ. Herein, we describe the experimental protocols and computational workflows used in our studies.

Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-ß-hydroxylase.

Aspartate/asparagine-ß-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.

Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies.

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.

In vitro assay and inhibition of 9-cis-epoxycarotenoid dioxygenase (NCED) from Solanum lycopersicum and Zea mays.

The article reports methods for the expression and assay of 9-cis-epoxycarotenoid cleavage dioxygenase (NCED), an enzyme involved in the biosynthesis of phytohormone abscisic acid in plants. A method for the preparation of the unstable substrate 9'-cis-neoxanthin from fresh spinach is described. The inhibition of Solanum lycopersicum NCED by a series of aryl hydroxamic acid inhibitors is illustrated, and inhibitors D2 and D4 are assayed against NCED isozymes from Zea mays.

Cyclopropanation and aziridination catalyzed by non-heme iron and 2-oxoglutarate dependent enzymes.

Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro.

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Best Practices for Development and Validation of Enzymatic Activity Assays to Support Drug Development for Inborn Errors of Metabolism and Biomarker Assessment.

Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management.

Fluorescent covalent organic framework as an ultrasensitive fluorescent probe for tyrosinase activity monitoring and inhibitor screening.

BACKGROUND: As a significant biomarker of melanocytic lesions, tyrosinase (TYR) plays an essential role in the clinical diagnosis and treatment of melanin-related diseases. Thus, it is important to develop robust methods for assessing TYR activity. Covalent organic frameworks (COFs) have garnered considerable attention owing to their unique properties, including high chemical stability, good biocompatibility, and large surface area compared with organic dyes, noble metal nanoclusters, and semiconductor quantum dots. However, most COFs are insoluble in water and exhibit weak or no fluorescence emission. Therefore, the development of a water-soluble fluorescent COF for detecting TYR activity in biological samples remains highly desired. RESULTS: In this work, a sensitive and facile fluorometric method based on fluorescent COF was constructed for the detection of TYR activity in human serum samples. The water-soluble COF was fabricated through the condensation polymerization of 4',4‴,4''''',4'''''''-(1,2-ethene-diylidene) tetrakis [1,1'-biphenyl]-4-carboxaldehyde and 2,4,6-tris-(4-aminophenyl)-triazine. The resulting COF displayed yellow-green fluorescence with a maximum emission peak at 560 nm. Tyrosine was catalyzed by TYR to produce melanin-like polymers which formed a coating on the surface of COF and effectively quenched its fluorescence due to fluorescence resonance energy transfer. The proposed approach demonstrated a strong linear correlation in the range of 0.5-80 U/L with a low detection limit of 0.09 U/L. Additionally, the limit of detection for kojic acid, serving as a representative TYR inhibitor, was determined to be 0.0004 µg/mL. SIGNIFICANCE: Our proposed fluorometric sensing platform exhibited exceptional selectivity, sensitivity, and satisfactory recoveries in human serum samples, which is of paramount importance for the clinical diagnostics of melanin-related diseases. Furthermore, the proposed approach was further employed for the screening of TYR inhibitors, suggesting the potential applications in clinical treatment and pharmaceutical research.

Methods for biochemical characterization of flavin-dependent N-monooxygenases involved in siderophore biosynthesis.

Siderophores are essential molecules released by some bacteria and fungi in iron-limiting environments to sequester ferric iron, satisfying metabolic needs. Flavin-dependent N-hydroxylating monooxygenases (NMOs) catalyze the hydroxylation of nitrogen atoms to generate important siderophore functional groups such as hydroxamates. It has been demonstrated that the function of NMOs is essential for virulence, implicating these enzymes as potential drug targets. This chapter aims to serve as a resource for the characterization of NMO's enzymatic activities using several biochemical techniques. We describe assays that allow for the determination of steady-state kinetic parameters, detection of hydroxylated amine products, measurement of the rate-limiting step(s), and the application toward drug discovery efforts. While not exhaustive, this chapter will provide a foundation for the characterization of enzymes involved in siderophore biosynthesis, allowing for gaps in knowledge within the field to be addressed.

Nanochannel-based biosensor for ultrasensitive and label-free detection of thymidine kinase activity.

Thymidine Kinase 1 (TK1) is a pivotal enzyme in fundamental biochemistry and molecular diagnosis, but recognition and molecule detection is a challenging task. Here, we constructed a DNA-integrated hybrid nanochannel sensor for TK1 activity and inhibition assay. Single-stranded DNA containing thymidine was used as a substrate to functionalize the nanochannels, restricting the ion current through channels. With kinase, the thymidine at the termini of the substrate DNA is phosphorylated, elevating surface charge density and mitigating the pore-obstruction effect by increasing transmembrane ion current. The kinase-induced distinctness can be accurately monitored by this hybrid nanodevice, which benefits from its high sensitivity to the change of surface charge. The excellent analytical performance in both kinase enzyme activity and inhibition analysis resulted in efficient and selective evaluation in human serum. Furthermore, compared to current approaches, it greatly simplifies and offers a direct method of analysis, making it a promising sensor technology for cancer management as well as the activities of multiple types of nucleic acid kinases.

In-line RNA-based microreactor direct mass spectrometry for ultrasensitive and rapid assay of terminal deoxynucleotidyl transferase activity.

Terminal deoxynucleotidyl transferase (TdT), a unique template-independent DNA polymerase, plays a crucial role in the human adaptive immune system and is considered a promising biomarker for the diagnosis of various forms of acute or chronic leukemia. The accurate and sensitive detection of trace TdT is of pivotal importance to fulfill the significant medical interest in understanding its pathological functions and diagnosing TdT-related diseases. We hereby present an in-line RNA-based microreactor direct mass spectrometry (MS) method and its application for ultrasensitive, accurate, and rapid analysis of trace TdT activity in leukemic cell samples. A specially designed RNA-based microreactor is fabricated by immobilizing short RNA sequence via covalent Au-S bond on the inner surface of a capillary pre-modified with three-dimensional porous layer (PL) and Au nanoparticles (AuNPs). Utilizing this PL@Au@RNA microreactor, the signal of target TdT is conversed into reporter molecules (adenine), which exhibit a strong MS response. This conversion process enables efficient signal amplification and enhances detection sensitivity. The outlet end of the PL@Au@RNA microreactor is deliberately crafted into a porous tip, serving as an electrospray ionization (ESI) interface to directly couple to ESI-MS in-line. This design facilitates the direct transmission of the generated signaling molecules into the MS system, eliminating the need for laborious sample treatment procedures. By implementing this RNA-based microreactor in direct MS analysis, we have achieved remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10-9 U, surpassing other reported methods in literature by three to four orders of magnitude. Furthermore, each assay requires a minimal sample volume of merely 10 nL. This method has successfully demonstrated its application in accurately and efficiently detecting TdT activity in leukemia cells, and its detection results are consistent with those obtained by ELISA kits.

4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

In Brassica plants, glucosinolates are a diverse class of natural products, of which aliphatic methionine-derived glucosinolates are the most abundant form. Their structural diversity comes from the elongation of some side-chains by up to 9 carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Methylthioalkylmalate synthase (MAMS) catalyzes the iterative elongation process for aliphatic methionine-derived glucosinolates. Most biochemical studies on MAMS have been performed using liquid chromatography/mass spectrometry (LC/MS)-based assays or high-performance liquid chromatography (HPLC)-based assays. The LC/MS- and HPLC-based methods are endpoint assays, which cannot be monitored in real time and require a laborious process for data collection. These analytical methods are inefficient for performing multiple enzymatic assays needed to determine steady-state kinetic parameters or for mechanistic evaluation of pH-dependence and kinetic isotope effect studies. Although the function of MAMS has long been defined, there is a gap in knowledge as it pertains to biochemical characterization of this plant enzyme. Part of this may be due to the lack of efficient methods that can be used for this type of research. This chapter describes a continuous photometric assay to track MAMS activity in real time using the 4-aldrithiol reagent for reaction detection.