One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse.

The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

Cysteine Enrichment Mediates Co-Option of Uricase in Reptilian Skin and Transition to Uricotelism.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with ß-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm-specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.

Identification of hidden associations among eukaryotic genes through statistical analysis of coevolutionary transitions.

Coevolution at the gene level, as reflected by correlated events of gene loss or gain, can be revealed by phylogenetic profile analysis. The optimal method and metric for comparing phylogenetic profiles, especially in eukaryotic genomes, are not yet established. Here, we describe a procedure suitable for large-scale analysis, which can reveal coevolution based on the assessment of the statistical significance of correlated presence/absence transitions between gene pairs. This metric can identify coevolution in profiles with low overall similarities and is not affected by similarities lacking coevolutionary information. We applied the procedure to a large collection of 60,912 orthologous gene groups (orthogroups) in 1,264 eukaryotic genomes extracted from OrthoDB. We found significant cotransition scores for 7,825 orthogroups associated in 2,401 coevolving modules linking known and unknown genes in protein complexes and biological pathways. To demonstrate the ability of the method to predict hidden gene associations, we validated through experiments the involvement of vertebrate malate synthase-like genes in the conversion of (S)-ureidoglycolate into glyoxylate and urea, the last step of purine catabolism. This identification explains the presence of glyoxylate cycle genes in metazoa and suggests an anaplerotic role of purine degradation in early eukaryotes.

A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking.

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249 -encoded transmembrane domain containing protein, CATSPERθ, is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper transmembrane subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might acts as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.

Expansion of nickel binding- and histidine-rich proteins during gastric adaptation of Helicobacter species.

Acquisition and homeostasis of essential metals during host colonization by bacterial pathogens rely on metal uptake, trafficking, and storage proteins. How these factors have evolved within bacterial pathogens is poorly defined. Urease, a nickel enzyme, is essential for Helicobacter pylori to colonize the acidic stomach. Our previous data suggest that acquisition of nickel transporters and a histidine-rich protein (HRP) involved in nickel storage in H. pylori and gastric Helicobacter spp. have been essential evolutionary events for gastric colonization. Using bioinformatics, proteomics, and phylogenetics, we extended this analysis to determine how evolution has framed the repertoire of HRPs among 39 Epsilonproteobacteria; 18 gastric and 11 non-gastric enterohepatic (EH) Helicobacter spp., as well as 10 other Epsilonproteobacteria. We identified a total of 213 HRPs distributed in 22 protein families named orthologous groups (OGs) with His-rich domains, including 15 newly described OGs. Gastric Helicobacter spp. are enriched in HRPs (7.7 ± 1.9 HRPs/strain) as compared to EH Helicobacter spp. (1.9 ± 1.0 HRPs/strain) with a particular prevalence of HRPs with C-terminal histidine-rich domains in gastric species. The expression and nickel-binding capacity of several HRPs was validated in five gastric Helicobacter spp. We established the evolutionary history of new HRP families, such as the periplasmic HP0721-like proteins and the HugZ-type heme oxygenases. The expansion of histidine-rich extensions in gastric Helicobacter spp. proteins is intriguing but can tentatively be associated with the presence of the urease nickel enzyme. We conclude that this HRP expansion is associated with unique properties of organisms that rely on large intracellular nickel amounts for their survival.

Origin and significance of the human DNase repertoire.

The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics and transcriptomics data to infer the evolutionary history of DNases and investigate their biological significance. Both DNase1 and DNase2 families have expanded in vertebrates since ~ 650 million years ago before the divergence of jawless and jawed vertebrates. DNase1, DNase1L1, and DNase1L3 co-existed in jawless fish, whereas DNase1L2 originated in amniotes by tandem duplication of DNase1. Among the non-human DNases, DNase1L4 and newly identified DNase1L5 derived from early duplications that were lost in terrestrial vertebrates. The ancestral gene of the DNase2 family, DNase2b, has been conserved in synteny with the Uox gene across 700 million years of animal evolution,while DNase2 originated in jawless fish. DNase1L1 acquired a GPI-anchor for plasma membrane attachment in bony fishes, and DNase1L3 acquired a C-terminal basic peptide for the degradation of microparticle DNA in jawed vertebrates. The appearance of DNase1L2, with a distinct low pH optimum and skin localization, is among the amniote adaptations to life on land. The expansion of the DNase repertoire in vertebrates meets the diversified demand for DNA debris removal in complex multicellular organisms.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

Author Correction: Female mouse tears contain an anti-aggression pheromone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Birth of a pathway for sulfur metabolism in early amniote evolution.

Among amniotes, reptiles and mammals are differently adapted to terrestrial life. It is generally appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo, but rather repurposing of existing ones. Here, by comparing the inventory of pyridoxal 5'-phosphate-dependent enzymes in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase contains haem and pyridoxal 5'-phosphate co-factors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide, respectively. A specific cysteic acid decarboxylase produces taurine, while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase. This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by cystathionine beta-synthase duplication, cysteine lyase neofunctionalization and cysteic acid decarboxylase co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways, and reveal the molecular mechanisms by which such innovations arose in amniote evolution.

Female mouse tears contain an anti-aggression pheromone.

Tears contain pheromones that trigger specific behavioral responses. In the mouse, male tear fluid is involved in long and short-term effects such as the receptive behavior and pregnancy block in females and the aggression in males. In contrast, pup tears exert an inhibitory effect on male mating behavior, also promoting sexual rejection in females. In the rat, a male lacrimal protein acts as an intraspecific and heterospecific signal enhancing sexual behavior in females and evoking avoidance behavior in mouse. However, behavioral effects of female tears on male behavior have yet to be described. Here, we report that female lacrimal fluid of different mouse strains contains a relatively small and involatile factor that abolishes inter-male aggression switching it into a copulatory behavior. The production of this molecule by the lacrimal glands is not affected by the estrous cycle but it is sensitive to ovariectomy, thus suggesting a control mediated by hormones. Moreover, this lacrimal anti-aggression pheromone modulates the activity of the lateral habenula, a brain area responsible for the valence of the aggressive interactions.

The peroxisomal SspA protein is redundant for purine utilization but essential for peroxisome localization in septal pores in Aspergillus nidulans.

In an in silico search for correlated gene loss with fungal peroxisomal uric acid oxidase (UOX), we identified PMP22-like proteins, some of which function as promiscuous channels in organellar membranes. To investigate whether PMP22 channels have a role in peroxisomal uric acid transport and catabolism, we functionally analyzed the closest homologue in Aspergillus nidulans, named SspA. We confirmed that SspA is a peroxisomal membrane protein that co-localizes significantly with PTS1-tagged mRFP, UOX or HexA, the latter considered a protein of Woronin bodies (WB), organelles originating from peroxisomes that dynamically plug septal pores in ascomycetes. Our results suggest that in A. nidulans, unlike some other ascomycetes, there is no strict protein segregation of peroxisomal and WB-specific proteins. Importantly, genetic deletion of sspA, but not of hexA, led to lack of peroxisomal localization at septal pores, suggesting that SspA is a key factor for septal pore functioning. Additionally, ΔsspA resulted in increased sensitivity to oxidative stress, apparently as a consequence of not only the inability to plug septal pores, but also a recorded reduction in peroxisome biogenesis. However, deleting sspA had no effect on uric acid or purine utilization, as we hypothesized, a result also in line with the observation that expression of SspA was not affected by regulatory mutants and conditions known to control purine catabolic enzymes. Our results are discussed within the framework of previous studies of SspA homologues in other fungi, as well as, the observed gene losses of PMP22 and peroxisomal uric acid oxidase.

Immobilization of Allantoinase for the Development of an Optical Biosensor of Oxidative Stress States.

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase-the enzyme responsible for allantoin hydrolization in plants and lower organisms-for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor.

Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP.

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs - but not SDHs - are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

Diatom Allantoin Synthase Provides Structural Insights into Natural Fusion Protein Therapeutics.

Humans have lost the ability to convert urate into the more soluble allantoin with the evolutionary inactivation of three enzymes of the uricolytic pathway. Restoration of this function through enzyme replacement therapy can treat severe hyperuricemia and Lesch-Nyhan disease. Through a genomic exploration of natural gene fusions, we found that plants and diatoms independently evolved a fusion protein (allantoin synthase) complementing two human pseudogenes. The 1.85-Å-resolution crystal structure of allantoin synthase from the diatom Phaeodactylum tricornutum provides a rationale for the domain combinations observed in the metabolic pathway, suggesting that quaternary structure is key to the evolutionary success of protein domain fusions. Polyethylene glycol (PEG) conjugation experiments indicate that a PEG-modified form of the natural fusion protein provides advantages over separate enzymes in terms of activity maintenance and manufacturing of the bioconjugate. These results suggest that the combination of different activities in a single molecular unit can simplify the production and chemical modification of recombinant proteins for multifunctional enzyme therapy.

Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus.

Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease.

PURPOSE: Because of the evolutionary loss of the uricolytic pathway, humans accumulate poorly soluble urate as the final product of purine catabolism. Restoration of uricolysis through enzyme therapy is a promising treatment for severe hyperuricemia caused by deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). To this end, we studied the effect of PEG conjugation on the activity and stability of the enzymatic complement required for conversion of urate into the more soluble (S)-allantoin. METHODS: We produced in recombinant form three zebrafish enzymes required in the uricolytic pathway. We carried out a systematic study of the effect of PEGylation on the function and stability of the three enzymes by varying PEG length, chemistry and degree of conjugation. We assayed in vitro the uricolytic activity of the PEGylated enzymatic triad. RESULTS: We defined conditions that allow PEGylated enzymes to retain native-like enzymatic activity even after lyophilization or prolonged storage. A combination of the three enzymes in an appropriate ratio allowed efficient conversion of urate to (S)-allantoin with no accumulation of intermediate metabolites. CONCLUSIONS: Pharmaceutical restoration of the uricolytic pathway is a viable approach for the treatment of severe hyperuricemia.

The renal phenotype of allopurinol-treated HPRT-deficient mouse.

Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 µg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems to be directly involved in promoting lipid accumulation and subsequent phenotype changes of tubular cells, with activation of inflammation and fibrosis.

[Recent advances in urate metabolism]. / Nuovi avanzamenti nelle conoscenze del metabolismo dell'urato.

In the last fifteen years, genomics and other -omics sciences have revolutionized our understanding of biological processes at the molecular level. An illustrative example is urate metabolism. Before the publication of the complete human genome, in 2003 it was believed that a single enzyme (urate oxidase) was responsible for uricolysis that is the conversion of urate into the more soluble allantoin. Now we know with great detail that this process requires the consecutive action of three enzymes that have been lost by gene inactivation in our hominoid ancestor. Similarly, a single urate transporter (URAT1) was known at that time. Now we have evidence that urate homeostasis depends on a complex set of transporters located on the epithelial cells of the kidney and the intestine. In this review article, we give an account of the recent discoveries on urate metabolism and how these discoveries can be applied to the development of novel drugs to treat hyperuricemia, tumor lysis syndrome and the Lesch-Nyhan disease.

Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids.

Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 µM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations.