The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.

A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.

Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.

Structure, biochemical and kinetic properties of recombinant Pst2p from Saccharomyces cerevisiae, a FMN-dependent NAD(P)H:quinone oxidoreductase.

The genome of the yeast Saccharomyces cerevisiae encodes four flavodoxin-like proteins, namely Lot6p, Pst2p, Rfs1p and Ycp4p. Thus far only Lot6p was characterized in detail demonstrating that the enzyme possesses NAD(P)H:quinone oxidoreductase activity. In the present study, we heterologously expressed PST2 in Escherichia coli and purified the produced protein to conduct a detailed biochemical and structural characterization. Determination of the three-dimensional structure by X-ray crystallography revealed that Pst2p adopts the flavodoxin-like fold and forms tetramers independent of cofactor binding. The lack of electron density for FMN indicated weak binding, which was confirmed by further biochemical analysis yielding a dissociation constant of 20±1µM. The redox potential of FMN bound to Pst2p was determined to -89±3mV and is thus 119mV more positive than that of free FMN indicating that reduced FMN binds ca. five orders of magnitude tighter to Pst2p than oxidized FMN. Due to this rather positive redox potential Pst2p is unable to reduce free FMN or azo dyes as reported for other members of the flavodoxin-like protein family. On the other hand, Pst2p efficiently catalyzes the NAD(P)H dependent two-electron reduction of natural and artificial quinones. The kinetic mechanism follows a ping-pong bi-bi reaction scheme. In vivo experiments with a PST2 knock out and overexpressing strain demonstrated that Pst2p enables yeast cells to cope with quinone-induced damage suggesting a role of the enzyme in managing oxidative stress.

Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution.

The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities-two histone marks and open chromatin state-from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3-4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development.

The scope of flavin-dependent reactions and processes in the model plant Arabidopsis thaliana.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are utilized as coenzymes in many biochemical reduction-oxidation reactions owing to the ability of the tricyclic isoalloxazine ring system to employ the oxidized, radical and reduced state. We have analyzed the genome of Arabidopsis thaliana to establish an inventory of genes encoding flavin-dependent enzymes (flavoenzymes) as a basis to explore the range of flavin-dependent biochemical reactions that occur in this model plant. Expectedly, flavoenzymes catalyze many pivotal reactions in primary catabolism, which are connected to the degradation of basic metabolites, such as fatty and amino acids as well as carbohydrates and purines. On the other hand, flavoenzymes play diverse roles in anabolic reactions most notably the biosynthesis of amino acids as well as the biosynthesis of pyrimidines and sterols. Importantly, the role of flavoenzymes goes much beyond these basic reactions and extends into pathways that are equally crucial for plant life, for example the production of natural products. In this context, we outline the participation of flavoenzymes in the biosynthesis and maintenance of cofactors, coenzymes and accessory plant pigments (e. g. carotenoids) as well as phytohormones. Moreover, several multigene families have emerged as important components of plant immunity, for example the family of berberine bridge enzyme-like enzymes, flavin-dependent monooxygenases and NADPH oxidases. Furthermore, the versatility of flavoenzymes is highlighted by their role in reactions leading to tRNA-modifications, chromatin regulation and cellular redox homeostasis. The favorable photochemical properties of the flavin chromophore are exploited by photoreceptors to govern crucial processes of plant adaptation and development. Finally, a sequence- and structure-based approach was undertaken to gain insight into the catalytic role of uncharacterized flavoenzymes indicating their involvement in unknown biochemical reactions and pathways in A. thaliana.

Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples.

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

A small molecule chaperone rescues the stability and activity of a cancer-associated variant of NAD(P)H:quinone oxidoreductase 1 in vitro.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD-dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single-nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline-based chemotherapy. In this study, we show that a small molecular chaperone (N-(2-bromophenyl)pyrrolidine-1-sulfonamide) repopulates the native wild-type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro.

Oxidative stress-induced structural changes in the microtubule-associated flavoenzyme Irc15p from Saccharomyces cerevisiae.

The genome of the yeast Saccharomyces cerevisiae encodes a canonical lipoamide dehydrogenase (Lpd1p) as part of the pyruvate dehydrogenase complex and a highly similar protein termed Irc15p (increased recombination centers 15). In contrast to Lpd1p, Irc15p lacks a pair of redox active cysteine residues required for the reduction of lipoamide and thus it is very unlikely that Irc15p performs a similar dithiol-disulfide exchange reaction as reported for lipoamide dehydrogenases. We expressed IRC15 in Escherichia coli and purified the produced protein to conduct a detailed biochemical characterization. Here, we show that Irc15p is a dimeric protein with one FAD per protomer. Photoreduction of the protein generates the fully reduced hydroquinone without the occurrence of a flavin semiquinone radical. Similarly, reduction with NADH or NADPH yields the flavin hydroquinone without the occurrence of intermediates as observed for lipoamide dehydrogenase. The redox potential of Irc15p was -313 ± 1 mV and is thus similar to lipoamide dehydrogenase. Reduced Irc15p is oxidized by several artificial electron acceptors such as potassium ferricyanide, 2,6-dichlorophenol-indophenol, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and menadione. However, disulfides such as cystine, glutathione, and lipoamide were unable to react with reduced Irc15p. Limited proteolysis and SAXS-measurements revealed that the NADH-dependent formation of hydrogen peroxide caused a substantial structural change in the dimeric protein. Therefore, we hypothesize that Irc15p undergoes a conformational change in the presence of elevated levels of hydrogen peroxide, which is a putative biomarker of oxidative stress. This conformational change may in turn modulate the interaction of Irc15p with other key players involved in regulating microtubule dynamics.

Catalytic competence, structure and stability of the cancer-associated R139W variant of the human NAD(P)H:quinone oxidoreductase 1 (NQO1).

The human NAD(P)H:quinone oxidoreductase 1 (NQO1; EC1.6.99.2) is an essential enzyme in the antioxidant defence system. Furthermore, NQO1 protects tumour suppressors like p53, p33ING1b and p73 from proteasomal degradation. The activity of NQO1 is also exploited in chemotherapy for the activation of quinone-based treatments. Various single nucleotide polymorphisms are known, such as NQO1*2 and NQO1*3 yielding protein variants of NQO1 with single amino acid replacements, i.e. P187S and R139W, respectively. While the former NOQ1 variant is linked to a higher risk for specific kinds of cancer, the role, if any, of the arginine 139 to tryptophan exchange in disease development remains obscure. On the other hand, mitomycin C-resistant human colon cancer cells were shown to harbour the NQO1*3 variant resulting in substantially reduced enzymatic activity. However, the molecular cause for this decrease remains unclear. In order to resolve this issue, recombinant NQO1 R139W has been characterized biochemically and structurally. In this report, we show by X-ray crystallography and 2D-NMR spectroscopy that this variant adopts the same structure both in the crystal as well as in solution. Furthermore, the kinetic parameters obtained for the variant are similar to those reported for the wild-type protein. Similarly, thermostability of the variant was only slightly affected by the amino acid replacement. Therefore, we conclude that the previously reported effects in human cancer cells cannot be attributed to protein stability or enzyme activity. Instead, it appears that loss of exon 4 during maturation of a large fraction of pre-mRNA is the major reason of the observed lack of enzyme activity and hence reduced activation of quinone-based chemotherapeutics.