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bioRxiv2024

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding (RBNS) reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions (IDR) flanking the ARID domain modulate the specificity and affinity of DNA-binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bi-functional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

biochemistry↗

bioRxiv2024

High-throughput determination of RNA tertiary contact thermodynamics by quantitative DMS chemical mapping

Structured RNAs often contain long-range tertiary contacts that are critical to their function. Despite the importance of tertiary contacts, methods to measure their thermodynamics are low throughput or require specialized instruments. Here, we introduce a new quantitative chemical mapping method (qDMS-MaPseq) to measure Mg2+-induced formation of tertiary contact thermodynamics in a high-throughput manner using standard biochemistry equipment. With qDMS-MaPseq, we measured the {Delta}G of 98 unique tetraloop/tetraloop receptor (TL/TLR) variants in a one-pot reaction. These results agree well with measurements from specialized instruments (R2=0.64). Furthermore, the DMS reactivity of the TL directly correlates to the stability of the contact (R2=0.68), the first direct evidence that DMS reactivity reports on thermodynamics. Combined with structure prediction, DMS reactivity allowed the development of experimentally accurate 3D models of TLR mutants. These results demonstrate that qDMS-MaPseq is broadly accessible, high-throughput, and directly links DMS reactivity to thermodynamics.

biochemistry↗

bioRxiv2024

Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni

Schistosomiasis is a parasitic disease caused by trematode worms of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. In this work we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni. We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while the levels of FMN increase. We show that live schistosomes can cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 {+/-} 5.9 {micro}M. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2 in the extracellular environment. Since schistosomes can be damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case, suggesting that covalently bound FAD on IL-4I1 is inaccessible to SmNPP5. We also report here that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 {+/-} 0.58 mM. Thus, the sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by, we hypothesize, the recently described schistosome riboflavin transporter SmaRT. In this work we also identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

biochemistry↗

bioRxiv2024

Human Saposin B Ligand Binding and Presentation to α-Galactosidase A

Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by -galactosidase A. However, the manner by which SapB stimulates -galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to -galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates -galactosidase A cleavage of Gb3-NBD in vitro. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactions between SapB and -galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to -galactosidase A. These findings establish general principles for molecular recognition in saposin:hydrolase complexes and highlight the utility of NBD reporter lipids in saposin biochemistry and structural biology.

biochemistry↗

bioRxiv2024

A look beyond the QR code of SNARE proteins

Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper-like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa-, Qb-, Qc-, and R-helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR-SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non-cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule.

biochemistry↗

bioRxiv2024

Cryo-EM Structure of HRSL Domain Reveals Activating Crossed Helices at the Core of GCN2

GCN2 is a conserved receptor kinase activating the Integrated Stress Response (ISR) in eukaryotic cells. The ISR kinases detect accumulation of stress molecules and reprogram translation from basal tasks to preferred production of cytoprotective proteins. GCN2 stands out evolutionarily among all protein kinases due to the presence of a histidyl tRNA synthetase-like (HRSL) domain, which arises only in GCN2 and is located next to the kinase domain. How HRSL contributes to GCN2 signaling remains unknown. Here we report a 3.2 [A] cryo-EM structure of HRSL from thermotolerant yeast Kluyveromyces marxianus. This structure shows a constitutive symmetrical homodimer featuring a compact helical-bundle structure at the junction between HRSL and kinase domains, in the core of the receptor. Mutagenesis demonstrates that this junction structure activates GCN2 and indicates that our cryo-EM structure captures the active signaling state of HRSL. Based on these results, we put forward a GCN2 regulation mechanism, where HRSL drives the formation of activated kinase dimers. Remaining domains of GCN2 have the opposite role and in the absence of stress they help keep GCN2 basally inactive. This autoinhibitory activity is relieved upon stress ligand binding. We propose that the opposing action of HRSL and additional GCN2 domains thus yields a regulated ISR receptor. Significance statementRegulation of protein synthesis (translation) is a central mechanism by which eukaryotic cells adapt to stressful conditions. In starving cells, this translational adaptation is achieved via the receptor kinase GCN2, which stays inactive under normal conditions, but is switched on under stress. The molecular mechanism of GCN2 switching is not well understood due to the presence of a structurally and biochemically uncharacterized histidyl tRNA synthetase-like domain (HRSL) at the core of GCN2. Here we use single-particle cryo-EM and biochemistry to elucidate the structure and function of HRSL. We identify a structure at the kinase/HRSL interface, which forms crossed helices and helps position GCN2 kinase domains for activation. These data clarify the molecular mechanism of GCN2 regulation.

biochemistry↗

bioRxiv2024

Functional Biosynthetic Stereodivergence in a Gene Cluster via a Dihydrosydnone N-oxide

Chirality features a critical role in the biochemistry of life and often only one enantiomeric series is observed (homochirality). Only few natural products have been obtained as racemates, e.g. the quorum-sensing signal valdiazen produced by Burkholderia cenocepacia H111. In this study, we investigated its biosynthetic gene cluster and discovered that both the enantiomerically pure (R)-fragin and the racemic valdiazen are obtained from the same pathway. This stereodivergence is based on the unusual heterocycle dihydrosydnone N-oxide intermediate, as evident from gene knockout, stable isotope feeding experiments, and mass spectrometry experiments. Both non-enzymatic racemisation via keto-enol tautomerisation and enzyme-mediated dynamic kinetic resolution were found to be crucial to this stereodivergent pathway. This novel mechanism underpins the production of configurationally and biologically distinct metabolites from a single gene cluster. Our findings highlight the intricate design of an intertwined biosynthesis pathway, providing a deeper understanding of microbial secondary metabolism related to microbial communication. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/591611v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@11b63corg.highwire.dtl.DTLVardef@d339e6org.highwire.dtl.DTLVardef@17e34b1org.highwire.dtl.DTLVardef@b58692_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Biochemical characterization of Bacillus anthracis sortase B: Use in sortase mediated ligation and substrate recognition dependent on residues beyond the canonical pentapeptide binding motif for sortase enzymes

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critical enzymes facilitate the attachment of proteins to the cell wall, and are potential targets for novel antibiotic development, as well as versatile tools in protein engineering applications. Although there are six classes of sortases recognized, class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes recognize the canonical Cell Wall Sorting Signal (CWSS), LPXTG, where X=any amino acid, although work in recent years identified additional promiscuity in multiple positions of this recognition motif. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes have previously been shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues at several positions. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. We also use chimeric proteins to identify a single site that improves baSrtB activity by [~]4-fold and use purified protein substrates to validate sortase-mediated ligation of two proteins using SrtB enzymes for the first time. Taken together, these studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.

biochemistry↗

bioRxiv2024

Cysteine S-acetylation is a post-translational modification involved in metabolic regulation

Cysteine is a reactive amino acid central to the catalytic activities of many enzymes. It is also a common target of post-translational modifications (PTMs), such as palmitoylation. This long-chain acyl PTM can modify cysteine residues and induce changes in protein subcellular localization. We hypothesized that cysteine could also be modified by short-chain acyl groups, such as cysteine S-acetylation. To test this, we developed sample preparation and non-targeted mass spectrometry protocols to analyze the mouse liver proteome for cysteine acetylation. Our findings revealed hundreds of sites of cysteine acetylation across multiple tissue types, revealing a previously uncharacterized cysteine acetylome. Cysteine acetylation shows a marked cytoplasmic subcellular localization signature, with tissue-specific acetylome patterns and specific changes upon metabolic stress. This study uncovers a novel aspect of cysteine biochemistry, highlighting short-chain modifications alongside known long-chain acyl PTMs. These findings enrich our understanding of the landscape of acyl modifications and suggest new research directions in enzyme activity regulation and cellular signaling in metabolism.

biochemistry↗

bioRxiv2024

The Interplay of Acetylation and Ubiquitination Controls PRMT1 Homeostasis

PRMT1 plays many important roles in both normal and disease biology, thus understanding its regulation is crucial. Herein, we report the role of p300-mediated acetylation at K228 in triggering PRMT1 degradation through FBXL17-mediated ubiquitination. Utilizing mass-spectrometry, cellular biochemistry, and genetic code-expansion technologies, we elucidate a crucial mechanism independent of PRMT1 transcript levels. These results underscore the significance of acetylation in governing protein stability and expand our understanding of PRMT1 homeostasis. By detailing the molecular interplay between acetylation and ubiquitination involved in PRMT1 degradation, this work contributes to broader efforts in deciphering post-translational mechanisms that influence protein homeostasis.

biochemistry↗

bioRxiv2024

Single and combined effect of salinity, heat, cold, and drought in Arabidopsis at metabolomics and photosynthetic levels

Ensuring food security is one of the main challenges related to a growing global population under climate change conditions. The increasing soil salinity levels, drought, heatwaves, and late chilling severely threaten crops and often co-occur in field conditions. This work aims to provide deeper insight into the impact of single vs combined abiotic stresses at the growth, biochemical and photosynthetic levels in Arabidopsis thaliana L. By studying single and combined stresses, stress interactions and synergic effects have been highlighted. Lower photosynthetic efficiency was recorded from the beginning in all the conditions that included salinity. Consistently, membrane stability and ROS production, combined with a targeted metabolomic quantification of glycine, GABA, proline, and glycine-betaine molecular markers, highlighted the hierarchically stronger impact of salinity and its combinations on plant biochemistry. Untargeted metabolomics coupled with multivariate statistics pointed out distinct metabolic reprogramming triggered by the different stress conditions, either alone or in combination, differentiating the impact of salinity, drought, and their combination with cold and heat. These results contribute to delving into the impact of various stress combinations, hierarchically highlighting the stress-specific effects and pointing out different interactions. HIGHLIGHTSCombined stresses highlighted synergic and stronger impact on Arabidopsis secondary metabolism, redox imbalance and photosynthetic performance compared to individual stresses. Overall, salinity and its combination were the most impactful.

biochemistry↗

bioRxiv2024

Structural basis of nucleotide selectivity in pyruvate kinase

Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism.

biochemistry↗

bioRxiv2024

Enzymatic carbon-fluorine bond cleavage by human gut microbes

Fluorinated compounds are used for agrochemical, pharmaceutical, and numerous industrial applications, resulting in global contamination. In many molecules, fluorine is incorporated to enhance the half-life and improve bioavailability. Fluorinated compounds enter the human body through food, water, and xenobiotics including pharmaceuticals, exposing gut microbes to these substances. The human gut microbiota is known for its xenobiotic biotransformation capabilities, but it was not previously known whether gut microbial enzymes could break carbon-fluorine bonds, potentially altering the toxicity of these compounds. Here, through the development of a rapid, miniaturized fluoride detection assay for whole-cell screening, we discovered active gut microbial defluorinases. We biochemically characterized enzymes from diverse human gut microbial classes including Clostridia, Bacilli, and Coriobacteriia, with the capacity to hydrolyze (di)fluorinated organic acids and a fluorinated amino acid. Whole-protein alanine scanning, molecular dynamics simulations, and chimeric protein design enabled the identification of a disordered C-terminal protein segment involved in defluorination activity. Domain swapping exclusively of the C-terminus conferred defluorination activity to a non-defluorinating dehalogenase. To advance our understanding of the structural and sequence differences between defluorinating and non-defluorinating dehalogenases, we trained machine learning models which identified protein termini as important features. Models trained on 41-amino acid segments from protein C-termini alone predicted defluorination activity with 83% accuracy (compared to 95% accuracy based on full-length protein features). This work is relevant for therapeutic interventions and environmental and human health by uncovering specificity-determining signatures of fluorine biochemistry from the gut microbiome. SignificanceHumans have introduced carbon-fluorine bonds into numerous manufactured compounds, including pharmaceuticals, leading to the formation of toxic fluorinated byproducts. While the human gut microbiota is known for its ability to metabolize drugs, its encoded capacity to break the strong carbon-fluorine chemical bond was previously unknown. Here we discovered that human gut microbial enzymes are capable of cleaving carbon-fluorine bonds. We developed a 96-well colorimetric fluoride assay amenable to bacterial culture-based screening. We additionally conducted whole-protein alanine scanning mutagenesis and identified through machine learning that flexible C-terminal loop residues were predictive of defluorination. Taken in the context of flexible regions of other enzyme families known to perform fluorine chemistry, this work supports using convergent structural features to predict defluorination specificity.

biochemistry↗

bioRxiv2024

Mechanistic basis of temperature-adaptation in microtubule dynamics across frog species

Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. We thus lack a mechanistic framework that links functional adaptability with environmental pressures. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures: we combine in vitro reconstitution assays, quantitative biochemistry, and cryogenic electron microscopy to show how a small number of primary sequence changes influences the energy landscape of tubulin interactions and thereby mediates cold-adaptation and microtubule stability. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches.

biochemistry↗

bioRxiv2024

Nonenzymatic, prebiotic aminoacylation couples chirality of RNA and protein

Life as we know it depends on the homochirality of nucleic acids and proteins. However, there is no widely accepted explanation for why life uses only D-sugars for nucleic acids and L-amino acids for proteins. Here we demonstrate a prebiotically plausible method of nonenzymatic aminoacylation in a water ice-eutectic phase. These reactions produce high yields of aminoacyl-tRNAs, which are active in translation. Surprisingly, we discovered these nonenzymatic aminoacylation conditions were stereoselective, favoring coupling of amino acids and RNA of "opposite" L- and D- configurations. D-RNA shows greater aminoacylation yields for L-amino acids. The opposite was true for L-RNA, which had greater yields with D-amino acids. Nucleic acid backbone chirality influencing stereoselectivity of aminoacylation presents the missing link in the origin of modern biochemistry. This phenomenon provides insight into the chirality of the RNA world, and helps to explain the "opposite" stereochemistry of modern biomolecules.

biochemistry↗

bioRxiv2024

The molecular mechanism of temperature-dependent phase separation of Heat shock factor 1

Heat shock factor 1 (HSF1) is the critical orchestrator for a cell responding to heat shock, and its dysfunction is linked to cancers and neurodegenerative diseases. HSF1 undergoes phase separation upon heat shock, and its activity is regulated by various post-translational modifications (PTMs). Despite its importance, the molecular details underlying HSF1 phase separation, temperature sensing, and post-translational modifications (PTMs) regulation remain poorly understood. Here, we discovered that HSF1 exhibits temperature-dependent phase separation with a lower critical solution temperature (LCST) behavior due to entropy contribution from solvent molecules, providing a new conceptual mechanism accounting for HSF1 activation. We employed a synergistic approach combining coarse-grain simulation and nuclear magnetic resonance spectroscopy to reveal the residue-level molecular details of the interactions driving the phase separation of wild-type HSF1 and its distinct PTM patterns at various temperatures. The identified interaction sites were further validated with biochemistry assays and mapped interface accounts for HSF1 functions reported. Importantly, the amino acid substitution experiment reveals the molecular grammar for temperature-dependent HSF1 phase separation is species-specific and physiologically relevant. These findings delineate chemical code that integrates protein PTM patterns with accurate phase separation for body physiological temperature control in animals.

biochemistry↗

bioRxiv2024

Protein identification using cryo-EM and artificial intelligence guides improved sample purification

Protein purification is essential in protein biochemistry, structural biology, and protein design. It enables the determination of protein structures, the study of biological mechanisms, and the biochemical and biophysical characterization of both natural and de novo designed proteins. Despite the broad application of various protein purification protocols, standard strategies can still encounter challenges, such as the unintended co-purification of unknown contaminants alongside the target protein. In particular, co-purification issues pose significant challenges for designed self-assembling protein nanomaterials, as it is difficult to determine whether unexpected observed geometries represent novel assembly states of the designed system, cross-contamination from other assemblies, or native proteins originating from the expression host. In this study, we assessed the ability of an automated structure-to-sequence pipeline to unambiguously identify an unknown co-purifying protein found across several purified designed protein samples. Using cryo-electron microscopy (Cryo-EM), ModelAngelos sequence-agnostic automated model-building feature, and the Basic Local Alignment Search Tool (BLAST), we identified the unknown protein as dihydrolipoamide succinyltransferase (DLST). This identification was further confirmed by comparing the cryo-EM data with available DLST structures in the Protein Data Bank (PDB) and AlphaFold 3 predictions from the top BLAST hits. The clear identification of DLST informed our subsequent literature search and led to the rational modification of our protein purification protocol, ultimately enabling the exclusion of the contaminant from preparations of our target nanoparticle. This study demonstrates the successful application of a structure-to-sequence workflow, integrating Cryo-EM, ModelAngelo, protein BLAST, PDB structures, and AlphaFold 3 predictions, to identify and remove an unknown protein from a purified sample. It also highlights the broader potential of integrating Cryo-EM with AI-driven tools for accurate protein identification across various samples and contexts within protein science. HighlightsO_LIAn unknown protein was consistently found in multiple de novo designed protein samples. C_LIO_LIThe protein was identified as dihydrolipoamide succinyltransferase (DLST) using Cryo-EM, ModelAngelo and BLAST, and further verified using AlphaFold 3 and the PDB. C_LIO_LIIdentification enabled rational modification of the purification protocol to exclude the contaminant. C_LIO_LIThis method enables accurate protein identification without requiring near-atomic resolution or prior sequence and structural data, making it broadly applicable to various areas of protein science. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/612515v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@592410org.highwire.dtl.DTLVardef@4ea3f3org.highwire.dtl.DTLVardef@edd0bcorg.highwire.dtl.DTLVardef@122fba2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Evolutionary origins and innovations sculpting the mammalian PRPS enzyme complex

The phosphoribosyl pyrophosphate synthetase (PRPS) enzyme conducts a chokepoint reaction connecting central carbon metabolism and nucleotide production pathways, making it essential for life1,2. Here, we show that the presence of multiple PRPS-encoding genes is a hallmark trait of eukaryotes, and we trace the evolutionary origins and define the individual functions of each of the five mammalian PRPS homologs - three isozymes (one testis-restricted)3,4 and two non-enzymatic associated proteins (APs)5,6 - which we demonstrate operate together as a large molecular weight complex capable of attaining a heterogeneous array of functional multimeric configurations. Employing a repertoire of isogenic fibroblast clones in all viable individual or combinatorial assembly states, we define preferential interactions between subunits, and we show that cells lacking PRPS2, PRPSAP1, and PRPSAP2 render PRPS1 into aberrant homo-oligomeric assemblies with diminished metabolic flux and impaired proliferative capacity. We demonstrate how numerous evolutionary innovations in the duplicated genes have created specialized roles for individual complex members and identify translational control mechanisms that enable fine-tuned regulation of PRPS assembly and activity, which provide clues into the positive and negative selective pressures that facilitate metabolic flexibility and tissue specialization in advanced lifeforms. Collectively, our study demonstrates how evolution has transformed a single PRPS gene into a multimeric complex endowed with functional and regulatory features that govern cellular biochemistry.

biochemistry↗