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bioRxiv2020

Effect of Increasing Glucose Concentration on Estimation of Electrolytes by ion selective electrode

BackgroundThe measurement of serum electrolytes like sodium (Na+), potassium (K+) is routinely performed in clinical biochemistry laboratories using ion-selective electrodes (ISE). AimTo minimize error and test the effect of increasing glucose concentration on estimation of sodium and potassium. Materials and MethodsRandomly selected sera samples submitted for routine biochemical estimations were pooled for the study. A stock solution of glucose with concentration of 20 g/ dL was prepared by dissolving anhydrous glucose in normal saline (0.9 % NaCl). Next the pooled sera was divided into several aliquots with 1.0 ml sera each and increasing amounts of the stock solution were added to the different aliquots so as to generate glucose concentrations ranging from around 100 mg/dL to about 3000 mg/dL. Resultsthe glucose concentration of different serum aliquots obtained on cobas c311(roche system) were found to rang from 110 to 2577 mg/dl. The mean of sodium by direct ion selective 142{+/-}0.52 mmol/l, values showed in significant difference (p=0.320).The mean of sodium by indirect ion selective 143 {+/-} 0.72 mmol/l, values showed significant difference (p< 0.05). And the mean of potassium by direct ion selective 3.54 {+/-}0.29 mmol/l, values showed significant difference (p< 0.05). The mean of potassium by indirect ion selective 3.53{+/-}0.26 mmol/l, values showed significant difference (p< 0.05). ConclusionsIncreasing blood gluose interferance in electrlytes mainly potasssium by both direct and indirect ion selective electrode, whenincreasing glucose potassium decreae significantly No different significant between direct and indirect ion selective electrods in potassium when used cobas c311 and easy lyte.Significant interferance in sodium by indirect ion selective electrodewhile no significant interferance in sodium direct ion selective electrode.Glucose concentration interfer the electrolyte specially potassium in concentration more than 1700 mg/dl by direct and indirect methods.

biochemistry↗

bioRxiv2021

Comparative and Stability Study of Glucose Concentrations Measured in Sodium Fluoride and Serum Separator tubes

IntroductionSodium fluoride/potassium oxalate (NaF/KOx) tubes were once regarded as the gold-standard tubes for glucose analysis. Even though their ineffectiveness in immediately inhibiting glycolysis has been reported in several studies especially in the first 1-4hours, they are still used in our clinical Biochemistry laboratory for glucose measurement. However in its absence, only SSTs are employed for glucose measurement. AimTo determine whether SSTs can replace NaF/KOx tubes for laboratory-based measurement of blood glucose and to assess the stability of glucose concentrations for 3 days period MethodsDuring the study period (1 March to 11 April, 2015), a total of 50 paired samples collected separately in NaF/KOx tubes and SSTs from healthy adult participants in the Gambia Adults Reference Intervals Study (GARIS) project were used as the project sample size. The samples were analysed within 2hours, and at 24hours, 42hours and 72hours time-points following blood collection and separation using Vitros 350 dry chemistry analyser. The GARIS samples were treated as clinical samples. ResultsThere was no significant difference in the mean glucose concentrations between the two tubes (Mean difference= 0.06mmol/L; P=0.38) recorded in the different time-points. Using growth trajectory and mixed effects model, the study data showed no significant change in the glucose concentrations (p=0.25) for three days period. ConclusionsThe study confirms that SSTs can produce similar glucose results when employed in the absence of NaF/KOx tubes. Besides, the glucose concentrations were stable in both tubes for three days when the samples were separated within two hours and refrigerated in 2-8{degrees}C.

biochemistry↗

bioRxiv2021

Mechanically Transduced Immunosorbent Assay To Measure Protein-Protein Interactions

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that indicates binding. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. Here we use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition.

biochemistry↗

bioRxiv2021

Topoisomerase VI is a chirally-selective, preferential DNA decatenase

DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase, however robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates, DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a dramatic increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90{degrees}.

biochemistry↗

bioRxiv2021

Interactome analysis of C. elegans synapses by TurboID-based proximity labeling

Proximity labelling provides a powerful in vivo tool to characterize the proteome of sub-cellular structures and the interactome of specific proteins. Using the highly active biotin ligase TurboID, we optimize a proximity labelling protocol for C. elegans. We use this protocol to characterise the proteomes of the worms gut, muscle, skin, and nervous system. We express TurboID exclusively in the pair of AFD neurons and show we can identify known and previously unknown proteins expressed selectively in AFD. We knock TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identify many known ELKS-1 interacting proteins as well as previously uncharacterised synaptic proteins. Versatile vectors, and the inherent advantage of C. elegans for biochemistry, make proximity labelling a valuable addition to the nematodes armory. TeaserWe optimize a TurboID proximity labeling protocol for C. elegans and use it to characterize tissue and synaptic proteomes

biochemistry↗

bioRxiv2021

An inventory of early branch points in microbial phosphonate biosynthesis

Microbial phosphonate biosynthetic machinery has been identified in ~5% of bacterial genomes and encodes natural products like fosfomycin as well as cell surface decorations. Almost all biological phosphonates originate from the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) catalyzed by PEP mutase (Ppm), and PnPy is often converted to phosphonoacetaldehyde (PnAA) by PnPy decarboxylase (Ppd). Seven enzymes are known or likely to act on either PnPy or PnAA as early branch points en route to diverse biosynthetic outcomes, and these enzymes may be broadly classified into three reaction types: hydride transfer, aminotransfer, and carbon-carbon bond formation. However, the relative abundance of these branch points in microbial phosphonate biosynthesis is unknown. Also unknown is the proportion of ppm-containing gene neighborhoods encoding new branch point enzymes and potentially novel phosphonates. In this study we computationally sorted 434 ppm-containing gene neighborhoods based on these seven branch point enzymes. Unsurprisingly, the majority (56%) of these pathways encode for production of the common naturally occurring compound 2-aminoethylphosphonate (AEP) or a hydroxylated derivative. The next most abundant genetically encoded intermediates were phosphonoalanine (PnAla, 9.2%), 2-hydroxyethylphosphonate (HEP, 8.5%), and phosphonoacetate (PnAc, 6%). Significantly, about 13% of the gene neighborhoods could not be assigned to any of the seven branch points and may encode novel phosphonates. Sequence similarity network analysis revealed families of unusual gene neighborhoods including possible production of phosphonoacrylate and phosphonofructose, the apparent biosynthetic use of the C-P lyase operon, and a virus-encoded phosphonate. Overall, these results highlight the utility of branch point inventories to identify novel gene neighborhoods and guide future phosphonate discovery efforts. IMPACT STATEMENTMicrobially-produced phosphonates are relatively rare and underexplored but include medically and agriculturally important molecules like fosfomycin and phosphinothricin, respectively. Because a single enzyme called phosphoenolpyruvate mutase (Ppm) inititates almost all phosphonate production, the composition of the gene neighborhood surrounding a Ppm-encoding gene can inform hypotheses regarding the chemical output of this chromosomal region. After the initial Ppm-catalyzed reaction there are only a limited set of subsequently acting enzymes, or branch points, to direct these early-stage phosphonates to alternate chemical fates. However, the relative abundance of different branch points - or the existence of new ones - has not been evaluated. This study provides just such a branch point inventory to determine relative proportions of known branch points and assess the diversity within each branch point. Significantly, this study suggests that a significant proportion (~13%) of gene neighborhoods do not fit into known branch points and therefore may be fertile hunting grounds for new phosphonate biochemistry. Data SummarySupporting information is available at Scholars Portal Dataverse (https://dataverse.scholarsportal.info/) with DOI 10.5683/SP2/T33ZP6. This includes scripts and the network data for visualizing in BiG-SCAPE and Cytoscape.

biochemistry↗

bioRxiv2021

Direct reconstitution and study of SUN protein interactions in vitro using mammalian cell-free expression

Membrane proteins perform numerous important functions in cells and tissues. Approximately 20% of the human genome encodes for membrane proteins, which represent the majority of targets for clinically relevant small molecules. Consequently, understanding their structure and structure-function relationships is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional membrane proteins in supported lipid bilayers. Despite having shown the directional reconstitution of CFE-synthesized FL inner nuclear membrane SUN proteins (i.e. SUN1 and SUN2), which directly interact with outer nuclear membrane KASH proteins within the nuclear envelope lumen to form linker of nucleoskeleton and cytoskeleton (LINC) complexes that mechanically couple the cytoskeleton and nucleus, the mechanism underlying regulated LINC complex assembly remains unclear. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment (e.g. calcium ions and redox state) on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and hetero-typic interactions that can occur between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies of membrane protein biochemistry and function, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins. ABSTRACT GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2021

Ca2+-mediated higher-order assembly of b0,+AT-rBAT is a key step for system b0,+ biogenesis and cystinuria

Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT-rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+ is the key to its trafficking on the cell surface. We show that Ca2+ stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+ site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+ site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.

biochemistry↗

bioRxiv2021

Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While {beta}-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.

biochemistry↗

bioRxiv2021

A dual allosteric pathway drives human mitochondrial Lon

The hexameric, barrel-forming, AAA+ protease Lon is critical for maintaining mitochondrial matrix protein homeostasis. Efficient substrate processing by Lon requires the coordinated action of six protomers. Despite Lons importance for human health, the molecular bases for Lons substrate recognition and processing remain unclear. Here, we use a combination of biochemistry and electron cryomicroscopy (cryo-EM) to unveil the structural and functional basis for full-length human mitochondrial Lons degradation of mitochondrial transcription factor A (TFAM). We show how opposing protomers in the Lon hexamer barrel interact through their N-terminal domains to give what resembles three feet above the barrel and help to form a triangular pore located just above the entry pore to the barrel. The interactions between opposing protomers constitute a primary allosteric regulation of Lon activity. A secondary allosteric regulation consists of an inter-subunit signaling element in the ATPase domains. By considering the ATP or ADP load in each protomer, we show how this dual allosteric mechanism in Lon achieves coordinated ATP hydrolysis and substrate processing. This mechanism enforces sequential anti-clockwise ATP hydrolysis resulting in a coordinated hand-over-hand translocation of the substrate towards the protease active sites.

biochemistry↗

bioRxiv2021

Rapid extraction and kinetic analysis of protein complexes from single cells

Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays, while being applicable to native protein complexes isolated from single cells in vivo.

biochemistry↗

bioRxiv2021

Implementing solid-phase-enhanced sample preparation for Co-Immunoprecipitation with Mass Spectrometry for C. elegans

Studying protein-protein interactions in vivo can reveal key molecular mechanisms of biological processes. Co-Immunoprecipitation followed by Mass Spectrometry (CoIP-MS) allows detection of protein-protein interactions in high-throughput. The nematode Caenorhabditis elegans (C. elegans) is a powerful genetic model organism for in vivo studies. Yet, its rigid cuticle and complex tissues require optimization for protein biochemistry applications to ensure robustness and reproducibility of experimental outcomes. Therefore, we optimized CoIP-MS application to C. elegans protein lysates by combining a native CoIP procedure with an efficient sample preparation method called single-pot, solid-phase-enhanced, sample preparation method (SP3). Our results based on the subunits of the conserved chromatin remodeler FACT demonstrate that our SP3-integrated CoIP-MS procedure for C. elegans samples is highly accurate and robust. Moreover, in a previous study (Baytek et al. 2021), we extended our technique to studying the chromodomain factor MRG-1 (MRG15 in human), which resulted in unprecedented findings. Method SummaryCombination of cryo-fracture with single-pot, solid-phase-enhanced, sample preparation (SP3) to perform Co-Immuno-Precipitation followed by Mass Spectrometry (CoIP-MS) provides robust assessments of protein-protein interaction using C. elegans whole animals.

biochemistry↗

bioRxiv2021

Development of affinity beads-based in vitro metal-ligand binding assay reveals dominant cadmium affinity of thiol-rich small peptides phytochelatins beyond glutathione

For a better understanding of metal-ligand interaction and its function in cells, we developed an easy, sensitive, and high-throughput method to quantify ligand-metal(loid) binding affinity under physiological conditions by combining ligand-attached affinity beads and inductively coupled plasma-optical emission spectrometry (ICP-OES). Glutathione (GSH) and two phytochelatins (PC2 and PC3, small peptides with different numbers of free thiols) were employed as model ligands and attached to hydrophilic beads. The principle of the assay resembles that of affinity purification of proteins in biochemistry: metals binding to the ligand on the beads and the rest in the buffer are separated by a spin-column and quantified by ICP-OES. The binding assay using the GSH-attached beads and various metal(loid)s suggested the different affinity of the metal-GSH interactions, in accordance with the order of the Irving-Williams series and the reported stability constants. The binding assay using PC2 or PC3-attached beads suggested positive binding between PCs and Ni(II), Cu(II), Zn(II), Cd(II), and As(III) in accordance with the number of thiols in PC2 and PC3. We then conducted the competition assay using Cd(II), Mn(II), Fe(II), Cu(II), and Zn(II) and the results suggested a better binding affinity of PC2 with Cd(II) than with the essential metals. Another competition assay using PC2 and GSH suggested a robust binding affinity between PCs and Cd(II) compared to GSH and Cd(II). These results suggested the dominance of PC-Cd complex formation in vitro, supporting the physiological importance of PCs for the detoxification of cadmium in vivo. We also discuss the potential application of the assay.

biochemistry↗

bioRxiv2021

Protein-Metabolite Interactomics Reveals Novel Regulation of Carbohydrate Metabolism

Metabolism is highly interconnected and also has profound effects on other cellular processes. However, the interactions between metabolites and proteins that mediate this connectivity are frequently low affinity and difficult to discover, hampering our understanding of this important area of cellular biochemistry. Therefore, we developed the MIDAS platform, which can identify protein-metabolite interactions with great sensitivity. We analyzed 33 enzymes from central carbon metabolism and identified 830 protein-metabolite interactions that were mostly novel, but also included known regulators, substrates, products and their analogs. We validated previously unknown interactions, including two atomic-resolution structures of novel protein-metabolite complexes. We also found that both ATP and long-chain fatty acyl-CoAs inhibit lactate dehydrogenase A (LDHA), but not LDHB, at physiological concentrations in vitro. Treating cells with long-chain fatty acids caused a loss of pyruvate/lactate interconversion, but only in cells reliant on LDHA. We propose that these regulatory mechanisms are part of the metabolic connectivity that enables survival in an ever-changing nutrient environment, and that MIDAS enables a broader and deeper understanding of that network.

biochemistry↗

bioRxiv2021

Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be useful when investigating the function and recognition of a broad range of disulfide-bond containing effectors.

biochemistry↗

bioRxiv2021

Semisynthesis of functional transmembrane proteins in GUVs

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

biochemistry↗

bioRxiv2021

A broad-specificity O-glycoprotease that enables improved analysis of glycoproteins and glycopeptides containing intact complex O-glycans

Analysis of mucin type O-glycans linked to serine/threonine of glycoproteins is technically challenging, in part, due to a lack of effective enzymatic tools that enable their analysis. Recently, several O-glycan-specific endoproteases that can cleave the protein adjacent to the appended glycan have been described. Despite significant progress in understanding the biochemistry of these enzymes, known O-glycoproteases have specificity constrains, such as inefficient cleavage of glycoproteins bearing sialylated O-glycans, high selectivity for certain type of glycoproteins or protein sequence bias, that limit their analytical application. In this study, we examined the capabilities of an immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa. The peptide substrate sequence selectivity and its impact on IMPa activity was interrogated using an array of synthetic peptides and their glycoforms. We show that IMPa has no specific P1 residue preference and can tolerate most amino acids at the P1 position, except aspartic acid. The enzyme does not cleave between two adjacent O-glycosites, indicating that O-glycosylated serine/threonine is not allowed at position P1. Glycopeptides with as few as two amino acids on either side of an O-glycosite were specifically cleaved by IMPa. Finally, IMPa efficiently cleaved peptides and proteins carrying sialylated and asialylated O-glycans of varying complexity. We present the use of IMPa in a one-step O-glycoproteomics workflow for glycoprofiling of individual purified glycoproteins granulocyte colony-stimulating factor (G-CSF) and receptor-type tyrosine-protein phosphatase C (CD45) without the need for glycopeptide enrichment. In these examples, IMPa enabled identification of O-glycosites and the range of complex O-glycan structures at each site.

biochemistry↗

bioRxiv2022

A novel, NADH-dependent acrylate reductase in Vibrio harveyi

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here we produced and characterized one such protein, Vibrio harveyi ARD, and found it to be a specific NADH:acrylate oxidoreductase. This previously unknown enzyme contains covalently bound FMN and non-covalently bound FAD and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to a threonine residue in the auxiliary FMN_bind domain. RT-qPCR and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically was high without acrylate and increased only twofold in its presence. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. ImportanceThe benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the ard gene product of V. harveyi (GenBank ID: AIV07243) exhibits a yet undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.

biochemistry↗