Publications by authors named "Riccardo Percudani"

50 Publications

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

Biomolecules 2021 Mar 10;11(3). Epub 2021 Mar 10.

Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124 Parma, Italy.

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.
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http://dx.doi.org/10.3390/biom11030410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002113PMC
March 2021

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

Sci Rep 2020 Nov 10;10(1):19805. Epub 2020 Nov 10.

Department of Medicine and Surgery, Neuroscience Unit, University of Parma, Via Volturno, 39, 43125, Parma, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-76734-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655806PMC
November 2020

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

Nat Ecol Evol 2020 09 29;4(9):1239-1246. Epub 2020 Jun 29.

Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parma, Italy.

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.
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http://dx.doi.org/10.1038/s41559-020-1232-4DOI Listing
September 2020

Female mouse tears contain an anti-aggression pheromone.

Sci Rep 2020 02 13;10(1):2510. Epub 2020 Feb 13.

Department of Medicine and Surgery, Neuroscience Unit, University of Parma, Via Volturno, 39, 43125, Parma, Italy.

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.
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http://dx.doi.org/10.1038/s41598-020-59293-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018997PMC
February 2020

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

Sensors (Basel) 2019 Dec 29;20(1). Epub 2019 Dec 29.

Centro Interdipartimentale Biopharmanet-TEC, Università di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy.

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.
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http://dx.doi.org/10.3390/s20010196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983136PMC
December 2019

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

Fungal Genet Biol 2019 11 5;132:103259. Epub 2019 Aug 5.

Department of Biology, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Athens, Greece. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fgb.2019.103259DOI Listing
November 2019

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

Sci Rep 2018 06 13;8(1):9016. Epub 2018 Jun 13.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università di Parma, Parma, Italy.

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.
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http://dx.doi.org/10.1038/s41598-018-27227-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998037PMC
June 2018

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

ACS Chem Biol 2018 08 28;13(8):2237-2246. Epub 2018 Jun 28.

Department of Chemistry, Life Sciences, and Environmental Sustainability , University of Parma , 43124 , Parma , Italy.

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.
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http://dx.doi.org/10.1021/acschembio.8b00404DOI Listing
August 2018

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

Sci Rep 2017 09 21;7(1):12051. Epub 2017 Sep 21.

Department of Food and Drug, University of Parma, 43124, Parma, Italy.

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.
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http://dx.doi.org/10.1038/s41598-017-12218-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608710PMC
September 2017

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

Pharm Res 2017 Jul 15;34(7):1477-1490. Epub 2017 May 15.

Department of Medicine and Surgery,, University of Parma, Parco Area delle Scienze 23/A, 43124, Parma, Italy.

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.
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http://dx.doi.org/10.1007/s11095-017-2167-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445154PMC
July 2017

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

PLoS One 2017 10;12(3):e0173512. Epub 2017 Mar 10.

Department of Life Sciences, University of Parma, Parma, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5345830PMC
August 2017

[Recent advances in urate metabolism].

G Ital Nefrol 2016 Malattie Metaboliche e Rene;33(S68)

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.
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October 2017

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

Sci Rep 2016 12 6;6:38302. Epub 2016 Dec 6.

Department of Life Sciences, University of Parma, 43124, Parma, Italy.

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.
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http://dx.doi.org/10.1038/srep38302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138847PMC
December 2016

Heme binding and peroxidase activity of a secreted minicatalase.

FEBS Lett 2016 Dec 28;590(24):4495-4506. Epub 2016 Nov 28.

Department of Life Sciences, University of Parma, Italy.

Microbial pathogens often require efficient and robust H O scavenger activities to survive in the presence of reactive oxygen species generated by inflammatory responses. In addition to catalases and peroxidases, enzymes known to scavenge H O , a novel class of secreted minicatalases is found in diderm bacteria. Here, we characterize the Helicobacter pylori (Hp) minicatalase: a monomeric hemoprotein with catalase core homology. Overexpression of Hp minicatalase rescued a catalase/peroxidase-deficient Escherichia coli phenotype under aerobic conditions and limited H O stress. The purified enzyme lacks catalase activity, but has strong (k > 100 s ) H O -dependent peroxidase activity toward a variety of organic substrates. Our investigations into heme binding revealed that the heme cofactor is assembled in the periplasm to form the functional holoprotein. Furthermore, we observed the presence of a disulfide bond near the heme cavity of Hp minicatalase, which is conserved in secreted minicatalases and, therefore, may play a role in heme binding.
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http://dx.doi.org/10.1002/1873-3468.12493DOI Listing
December 2016

The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism.

Biochemistry 2016 Nov 7;55(46):6421-6432. Epub 2016 Nov 7.

Department of Life Sciences, University of Parma , Parma, Italy.

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (k/K ≈ 5 × 10 M s) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by C and H nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution and conserved in spite of the substitution of catalytic residues.
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http://dx.doi.org/10.1021/acs.biochem.6b00881DOI Listing
November 2016

Correction: Absolute stereochemistry and preferred conformations of urate degradation intermediates from computed and experimental circular dichroism spectra.

Org Biomol Chem 2016 Apr 16;14(14):3654. Epub 2016 Mar 16.

Dipartimento di Chimica "G.I.A.F", Universitá di Parma, 43100, Parma, Italy.

Correction for 'Absolute stereochemistry and preferred conformations of urate degradation intermediates from computed and experimental circular dichroism spectra' by Silvio Pipolo et al., Org. Biomol. Chem., 2011, 9, 5149-5155.
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http://dx.doi.org/10.1039/c6ob90042aDOI Listing
April 2016

The identification of an integral membrane, cytochrome c urate oxidase completes the catalytic repertoire of a therapeutic enzyme.

Sci Rep 2015 Sep 8;5:13798. Epub 2015 Sep 8.

Department of Life Sciences, University of Parma, Italy.

In living organisms, the conversion of urate into allantoin requires three consecutive enzymes. The pathway was lost in hominid, predisposing humans to hyperuricemia and gout. Among other species, the genomic distribution of the two last enzymes of the pathway is wider than that of urate oxidase (Uox), suggesting the presence of unknown genes encoding Uox. Here we combine gene network analysis with association rule learning to identify the missing urate oxidase. In contrast with the known soluble Uox, the identified gene (puuD) encodes a membrane protein with a C-terminal cytochrome c. The 8-helix transmembrane domain corresponds to DUF989, a family without similarity to known proteins. Gene deletion in a PuuD-encoding organism (Agrobacterium fabrum) abolished urate degradation capacity; the phenotype was fully restored by complementation with a cytosolic Uox from zebrafish. Consistent with H2O2 production by zfUox, urate oxidation in the complemented strain caused a four-fold increase of catalase. No increase was observed in the wild-type, suggesting that urate oxidation by PuuD proceeds through cytochrome c-mediated electron transfer. These findings identify a missing link in purine catabolism, assign a biochemical activity to a domain of unknown function (DUF989), and complete the catalytic repertoire of an enzyme useful for human therapy.
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http://dx.doi.org/10.1038/srep13798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562309PMC
September 2015

Evolution of the Selenoproteome in Helicobacter pylori and Epsilonproteobacteria.

Genome Biol Evol 2015 Sep 4;7(9):2692-704. Epub 2015 Sep 4.

Department of Life Sciences, University of Parma, Italy

By competing for the acquisition of essential nutrients, Helicobacter pylori has the unique ability to persist in the human stomach, also causing nutritional insufficiencies in the host. Although the H. pylori genome apparently encodes selenocysteine synthase (SelA, HP1513), a key pyridoxal phosphate (PLP)-dependent enzyme for the incorporation of selenium into bacterial proteins, nothing is known about the use of this essential element in protein synthesis by this pathogen. We analyzed the evolution of the complete machinery for incorporation of selenium into proteins and the selenoproteome of several H. pylori strains and related Epsilonproteobacteria. Our searches identified the presence of selenoproteins-including the previously unknown DUF466 family-in various Epsilonproteobacteria, but not in H. pylori. We found that a complete system for selenocysteine incorporation was present in the Helicobacteriaceae ancestor and has been recently lost before the split of Helicobacter acinonychis and H. pylori. Our results indicate that H. pylori, at variance with other gastric and enterohepatic Helicobacter, does not use selenocysteine in protein synthesis and does not use selenium for tRNA wobble base modification. However, selA has survived as a functional gene, having lost the domain for the binding of selenocysteine tRNA, but maintaining the ability to bind the PLP cofactor. The evolutionary modifications described for the SelA protein of H. pylori find parallels in other bacterial and archaeal species, suggesting that an alternative enzymatic function is hidden in many proteins annotated as selenocysteinyl-tRNA synthase.
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http://dx.doi.org/10.1093/gbe/evv177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607533PMC
September 2015

The crystal structure of Helicobacter pylori HP1029 highlights the functional diversity of the sialic acid-related DUF386 family.

FEBS J 2015 Sep 1;282(17):3311-22. Epub 2015 Jul 1.

Department of Biomedical Sciences, University of Padua, Italy.

The proteins of the YhcH/YjgK/YiaL (DUF386) family have been implicated in the bacterial metabolism of host-derived sialic acids and biofilm formation, although their precise biochemical function remains enigmatic. We present here the crystal structure of protein HP1029 from Helicobacter pylori. The protein is a homodimer, in which each monomer comprises a molecular core formed by 12 antiparallel β-strands arranged in two β-sheets flanked by helices. The sandwich formed by the sheets assumes the shape of a funnel opened at one end, with a zinc ion present at the bottom of the funnel. The crystal structure unequivocally shows that HP1029 belongs to the DUF386 family. Although no bioinformatics evidence has been found for sialic acid catabolism in H. pylori, the genomic context of HP1029 in Helicobacter and related organisms suggests a possible role in the metabolism of bacterial surface saccharides, such as pseudaminic acid and its derivatives.
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http://dx.doi.org/10.1111/febs.13344DOI Listing
September 2015

Evolution of spatially coexpressed families of type-2 vomeronasal receptors in rodents.

Genome Biol Evol 2014 Dec 23;7(1):272-85. Epub 2014 Dec 23.

Department of Neuroscience, University of Parma, Italy

The vomeronasal organ (VNO) is an olfactory structure for the detection of pheromones. VNO neurons express three groups of unrelated G-protein-coupled receptors. Type-2 vomeronasal receptors (V2Rs) are specifically localized in the basal neurons of the VNO and are believed to sense protein pheromones eliciting specific reproductive behaviors. In murine species, V2Rs are organized into four families. Family-ABD V2Rs are expressed monogenically and coexpress with family-C V2Rs of either subfamily C1 (V2RC1) or subfamily C2 (V2RC2), according to a coordinate temporal diagram. Neurons expressing the phylogenetically ancient V2RC1 coexpress family-BD V2Rs or a specific group of subfamily-A V2Rs (V2RA8-10), whereas a second neuronal subset (V2RC2-positive) coexpresses a recently expanded group of five subfamily-A V2Rs (V2RA1-5) along with vomeronasal-specific Major Histocompatibility Complex molecules (H2-Mv). Through database mining and Sanger sequencing, we have analyzed the onset, diversification, and expansion of the V2R-families throughout the phylogeny of Rodentia. Our results suggest that the separation of V2RC1 and V2RC2 occurred in a Cricetidae ancestor in coincidence with the evolution of the H2-Mv genes; this phylogenetic event did not correspond with the origin of the coexpressing V2RA1-5 genes, which dates back to an ancestral myomorphan lineage. Interestingly, the evolution of receptors within the V2RA1-5 group may be implicated in the origin and diversification of some of the V2R putative cognate ligands, the exocrine secreting peptides. The establishment of V2RC2, which probably reflects the complex expansion and diversification of family-A V2Rs, generated receptors that have probably acquired a more subtle functional specificity.
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http://dx.doi.org/10.1093/gbe/evu283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4316634PMC
December 2014

Gene context analysis reveals functional divergence between hypothetically equivalent enzymes of the purine-ureide pathway.

Biochemistry 2014 Feb 23;53(4):735-45. Epub 2014 Jan 23.

Laboratory of Biochemistry, Molecular Biology, and Bioinformatics, Department of Life Sciences, University of Parma , Italy.

A major problem of genome annotation is the assignment of a function to a large number of genes of known sequences through comparison with a relatively small number of experimentally characterized genes. Because functional divergence is a widespread phenomenon in gene evolution, the transfer of a function to homologous genes is not a trivial exercise. Here, we show that a family of homologous genes which are found in purine catabolism clusters and have hypothetically equivalent functions can be divided into two distinct groups based on the genomic distribution of functionally related genes. One group (UGLYAH) encodes proteins that are able to release ammonia from (S)-ureidoglycine, the enzymatic product of allantoate amidohydrolase (AAH), but are unable to degrade allantoate. The presence of a gene encoding UGLYAH implies the presence of AAH in the same genome. The other group (UGLYAH2) encodes proteins that are able to release ammonia from (S)-ureidoglycine as well as urea from allantoate. The presence of a gene encoding UGLYAH2 implies the absence of AAH in the same genome. Because (S)-ureidoglycine is an unstable compound that is only formed by the AAH reaction, the in vivo function of this group of enzymes must be the release of urea from allantoate (allantoicase activity), while ammonia release from (S)-ureidoglycine is an accessory activity that evolved as a specialized function in a group of genes in which the coexistence with AAH was established. Insights on the active site modifications leading to a change in the enzyme activity were provided by comparison of three-dimensional structures of proteins belonging to the two different groups and by site-directed mutagenesis. Our results indicate that when the neighborhood of uncharacterized genes suggests a role in the same process or pathway of a characterized homologue, a detailed analysis of the gene context is required for the transfer of functional annotations.
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http://dx.doi.org/10.1021/bi4010107DOI Listing
February 2014

Ureidoglycolate hydrolase, amidohydrolase, lyase: how errors in biological databases are incorporated in scientific papers and vice versa.

Database (Oxford) 2013 9;2013:bat071. Epub 2013 Oct 9.

Department of Life Sciences, Laboratory of Biochemistry, Molecular Biology and Bioinformatics, University of Parma, Italy.

An opaque biochemical definition, an insufficient functional characterization, an interpolated database description, and a beautiful 3D structure with a wrong reaction. All these are elements of an exemplar case of misannotation in biological databases and confusion in the scientific literature concerning genes and enzymes acting on ureidoglycolate, an intermediate of purine catabolism. Here we show biochemical evidence for the relocation of genes assigned to EC 3.5.3.19 (ureidoglycolate hydrolase, releasing ammonia), such as allA of Escherichia coli or DAL3 of Saccharomyces cerevisiae, to EC 4.3.2.3 (ureidoglycolate lyase, releasing urea). The EC 3.5.3.19 should be more appropriately named ureidoglycolate amidohydrolase and include genes equivalent to UAH of Arabidopsis thaliana. The distinction between ammonia- or urea-releasing activities from ureidoglycolate is relevant for the understanding of nitrogen metabolism in various organisms and of virulence factors in certain pathogens rather than a nomenclature problem. We trace the original fault in database annotation and provide a rationale for its incorporation and persistence in the scientific literature. Notwithstanding the technological distance, yet not surprising for the constancy of human nature, error categories and mechanisms established in the study of the work of amanuensis monks still apply to the modern curation of biological databases.
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http://dx.doi.org/10.1093/database/bat071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793230PMC
March 2014

A Microbial Metagenome (Leucobacter sp.) in Caenorhabditis Whole Genome Sequences.

Bioinform Biol Insights 2013 24;7:55-72. Epub 2013 Feb 24.

Department of Biosciences, Laboratory of Biochemistry and Molecular Biology, University of Parma, Parma, Italy.

DNA of apparently recent bacterial origin is found in the genomic sequences of Caenorhabditis angaria and Caenorhabditis remanei. Here we present evidence that the DNA belongs to a single species of the genus Leucobacter (high-GC Gram+ Actinobacteria). Metagenomic tools enabled the assembly of the contaminating sequences in a draft genome of 3.2 Mb harboring 2,826 genes. This information provides insight into a microbial organism intimately associated with Caenorhabditis as well as a solid basis for the reassignment of 3,373 metazoan entries of the public database to a novel bacterial species (Leucobacter sp. AEAR). The application of metagenomic techniques can thus prevent annotation errors and reveal unexpected genetic information in data obtained by conventional genomics.
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http://dx.doi.org/10.4137/BBI.S11064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583267PMC
April 2013

Structural and functional insights into (S)-ureidoglycine aminohydrolase, key enzyme of purine catabolism in Arabidopsis thaliana.

J Biol Chem 2012 May 5;287(22):18796-805. Epub 2012 Apr 5.

Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

The ureide pathway has recently been identified as the metabolic route of purine catabolism in plants and some bacteria. In this pathway, uric acid, which is a major product of the early stage of purine catabolism, is degraded into glyoxylate and ammonia via stepwise reactions of seven different enzymes. Therefore, the pathway has a possible physiological role in mobilization of purine ring nitrogen for further assimilation. (S)-Ureidoglycine aminohydrolase enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, we report a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH). The crystal structure of AtUGlyAH in the ligand-free form shows a monomer structure in the bicupin fold of the β-barrel and an octameric functional unit as well as a Mn(2+) ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn(2+) ion acts as a molecular anchor to bind (S)-ureidoglycine, and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn(2+) ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism.
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http://dx.doi.org/10.1074/jbc.M111.331819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365701PMC
May 2012

A recent class of chemosensory neurons developed in mouse and rat.

PLoS One 2011 9;6(9):e24462. Epub 2011 Sep 9.

Department of Neuroscience, University of Parma, Parma, Italy.

In most animal species, the vomeronasal organ ensures the individual recognition of conspecifics, a prerequisite for a successful reproduction. The vomeronasal organ expresses several receptors for pheromone detection. Mouse vomeronasal type-2 receptors (V2Rs) are restricted to the basal neurons of this organ and organized in four families. Family-A, B and D (family ABD) V2Rs are expressed monogenically (one receptor per neuron) and coexpress with either Vmn2r1 or Vmn2r2, two members of family-C V2Rs. Thus, basal neurons are characterized by specific combinations of two V2Rs. To investigate this issue, we raised antibodies against all family-C V2Rs and analyzed their expression pattern. We found that six out of seven family-C V2Rs (Vmn2r2-7) largely coexpressed and that none of the anti-Vmn2r2-7 antibodies significantly stained Vmn2r1 positive neurons. Thus, basal neurons are divided into two complementary subsets. The first subset (Vmn2r1-positive) preferentially coexpresses a distinct group of family-ABD V2Rs, whereas the second subset (Vmn2r2-7-positive) coexpresses the remaining group of V2Rs. Phylogenetic reconstruction and the analysis of genetic loci in various species reveal that receptors expressed by this second neuronal subset are recent branches of the V2R tree exclusively present in mouse and rat. Conversely, V2Rs expressed in Vmn2r1 positive neurons, are phylogenetically ancient and found in most vertebrates including rodents. Noticeably, the more recent neuronal subset expresses a type of Major Histocompatibility Complex genes only found in murine species. These results indicate that the expansion of the V2R repertoire in a murine ancestor occurred with the establishment of a new population of vomeronasal neurons in which coexists the polygenic expression of a recent group of family-C V2Rs (Vmn2r2-7) and the monogenic expression of a recent group of family-ABD V2Rs. This evolutionary innovation could provide a molecular rationale for the exquisite ability in individual recognition and mate choice of murine species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024462PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170373PMC
June 2012

Absolute stereochemistry and preferred conformations of urate degradation intermediates from computed and experimental circular dichroism spectra.

Org Biomol Chem 2011 Jul 7;9(14):5149-55. Epub 2011 Jun 7.

Dipartimento di Chimica G.I.A.F, Universitá di Parma, 43100, Parma, Italy.

The enzymatic oxidation of urate leads to the sequential formation of optically active intermediates with unknown stereochemistry: (-)-5-hydroxyisourate (HIU) and (-)-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU). In accordance with the observation that a defect in HIU hydrolase causes hepatocarcinoma in mouse, a detoxification role has been proposed for the enzymes accelerating the conversion of HIU and OHCU into optically active (+)-allantoin. The enzymatic products of urate oxidation are normally not present in humans, but are formed in patients treated with urate oxidase. We used time-dependent density functional theory (TDDFT) to compute the electronic circular dichroism (ECD) spectra of the chiral compounds of urate degradation (HIU, OHCU, allantoin) and we compared the results with experimentally measured ECD spectra. The calculated ECD spectra for (S)-HIU and (S)-OHCU reproduced well the experimental spectra obtained through the enzymatic degradation of urate. Less conclusive results were obtained with allantoin, although the computed optical rotations in the transparent region supported the original assignment of the (+)-S configuration. These absolute configuration assignments can facilitate the study of the enzymes involved in urate metabolism and help us to understand the mechanism leading to the toxicity of urate oxidation products.
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http://dx.doi.org/10.1039/c1ob05433cDOI Listing
July 2011

The structure of Helicobacter pylori HP0310 reveals an atypical peptidoglycan deacetylase.

PLoS One 2011 Apr 29;6(4):e19207. Epub 2011 Apr 29.

Department of Biological Chemistry, University of Padua, Padua, Italy.

Peptidoglycan deacetlyase (HP0310, HpPgdA) from the gram-negative pathogen Helicobacter pylori, has been indicated as the enzyme responsible for a peptidoglycan modification that counteracts the host immune response. HpPgdA has been cloned, purified and expressed in good yield in E. coli. It has been crystallized, its structure determined and activity tests in vitro performed. The enzyme, which belongs to the polysaccharide deacetylases protein family, is a homo-tetramer. The four polypeptide chains, each folded into a single domain characterized by a non-canonical TIM-barrel fold, are arranged around a four-fold symmetry axis. The active site, one per monomer, contains a heavy ion coordinated in a way similar to other deacetylases. However, the enzyme showed no in vitro activity on the typical polysaccharide substrates of peptidoglycan deacetylases. In striking contrast with the known peptidoglycan deacetylases, HpPgdA does not exhibit a solvent-accessible polysaccharide binding groove, suggesting that the enzyme binds a small molecule at the active site.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019207PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084791PMC
April 2011

Probing the evolution of hydroxyisourate hydrolase into transthyretin through active-site redesign.

J Mol Biol 2011 Jun 16;409(4):504-12. Epub 2011 Apr 16.

Department of Biological Chemistry, University of Padua, Viale Colombo 3, 35121 Padua, Italy.

5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins.
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http://dx.doi.org/10.1016/j.jmb.2011.04.022DOI Listing
June 2011

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III.

Nucleic Acids Res 2011 Jul 17;39(13):5499-512. Epub 2011 Mar 17.

Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.
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http://dx.doi.org/10.1093/nar/gkr074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141230PMC
July 2011

An aminotransferase branch point connects purine catabolism to amino acid recycling.

Nat Chem Biol 2010 Nov 19;6(11):801-6. Epub 2010 Sep 19.

Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy.

Although amino acids are known precursors of purines, a pathway for the direct recycling of amino acids from purines has never been described at the molecular level. We provide NMR and crystallographic evidence that the PucG protein from Bacillus subtilis catalyzes the transamination between an unstable intermediate ((S)-ureidoglycine) and the end product of purine catabolism (glyoxylate) to yield oxalurate and glycine. This activity enables soil and gut bacteria to use the animal purine waste as a source of carbon and nitrogen. The reaction catalyzed by (S)-ureidoglycine-glyoxylate aminotransferase (UGXT) illustrates a transamination sequence in which the same substrate provides both the amino group donor and, via its spontaneous decay, the amino group acceptor. Structural comparison and mutational analysis suggest a molecular rationale for the functional divergence between UGXT and peroxisomal alanine-glyoxylate aminotransferase, a fundamental enzyme for glyoxylate detoxification in humans.
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http://dx.doi.org/10.1038/nchembio.445DOI Listing
November 2010