Riccardo Percudani

A genomic overview of pyridoxal‐phosphate‐dependent enzymes

Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free‐living prokaryotes, almost 1.5% of all genes code for PLP‐dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP‐dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP‐dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP‐dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP‐dependent activities than it has genes for PLP‐dependent enzymes. This observation presumably applies to many other classes of protein‐encoding genes.

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

A nutrient‐regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

Important morphogenetic transitions in fungi are triggered by starvation‐induced changes in the expression of structural surface proteins. Here, we report that nutrient deprivation causes a strong and reversible up‐regulation of TbSP1, a surface‐associated, Ca2+‐dependent phospholipase from the mycorrhizal fungus Tuber borchii. TbSP1 is the first phospholipase A2 to be described in fungi and identifies a novel class of phospholipid‐hydrolyzing enzymes. The TbSP1 phospholipase, which is synthesized initially as a pre‐protein, is processed efficiently and secreted during the mycelial phase. The mature protein, however, also localizes to the inner cell wall layer, close to the plasma membrane, in both free‐living and symbiosis‐engaged hyphae. It thus appears that a dual localization phospholipase A2 is involved in the adaptation of a symbiotic fungus to conditions of persistent nutritional limitation. Moreover, the fact that TbSP1‐related sequences are present in Streptomyces and Neurospora, and not in wholly sequenced non‐filamentous microorganisms, points to a general role for TbSP1 phospholipases A2 in the organization of multicellular filamentous structures in bacteria and fungi.

A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii☆

An ammonium transporter cDNA, named TbAMT1, was isolated from the ectomycorrhizal ascomycetous truffle Tuber borchii. The polypeptide encoded by TbAMT1 (52 kDa) functionally complements ammonium uptake-defective yeast mutants and shares sequence similarity with previously characterized ammonium transporters from Saccharomyces (Mep) and Arabidopsis (AtAMT1). Structural characteristics common to the Mep/Amt family and peculiar features of the Tuber transporter have been evidenced by a detailed topological model of the TbAMT1 protein, which predicts 11 transmembrane helices with an N terminus(OUT)/C terminus(IN) orientation. As revealed by uptake/competition experiments conducted in yeast, TbAMT1 is a high-affinity transporter with an apparent K(m) for ammonium of 2 microM. The TbAMT1 mRNA was very slowly, yet specifically upregulated in nitrogen-deprived T. borchii mycelia. Instead, a much faster return to basal expression levels was observed upon resupplementation of either ammonium or nitrate, which thus appear to be utilized as equally effective nitrogen sources by Tuber mycelia.

The anti-HIV cyanovirin-N domain is evolutionarily conserved and occurs as a protein module in eukaryotes

A novel protein family homologous to the sugar‐binding antiviral protein cyanovirin‐N (CVN) is described. CVN, an 11‐kDa protein that, by binding to the high‐mannose moiety of certain viral surface glycoproteins, blocks virus entry into target cells, has thus far been identified only in the cyanobacterium Nostoc ellipsosporum. Here we show that CVN belongs to a protein family identified by analysis of transcript sequences deriving from a gene expression profiling study conducted in the truffle Tuber borchii. Members of this family (named CyanoVirin‐N Homology) are found in filamentous ascomycetes and in the fern Ceratopteris richardii. As revealed by 3D structure‐based searches, all CVNH proteins have a predicted fold that matches the so far unique fold of the cyanobacterial polypeptide. The CVNH domain is a versatile protein module. In ferns and cyanobacteria it is found in secretory proteins. In filamentous ascomycetes it is found in nonsecretory monodomain proteins as well as part of multidomain proteins bearing functionally related modules such as the peptidoglycan and chitin‐binding domain LysM. Transcript abundance data further indicate that the expression of different CVNH forms is modulated in response to nutrient availability. These findings have implications for the understanding of protein–oligosaccharide interaction in fungi and plants, and provide candidate polypeptides to be tested and exploited as antiviral agents. Proteins 2005.

Conserved Alternative Splicing of Arabidopsis Transthyretin-Like Determines Protein Localization and S-Allantoin Synthesis in Peroxisomes

Conserved alternative splicing of an unusual internal targeting sequence turns the enzyme completing the peroxisomal pathway of ureide biosynthesis into a cytosolic protein with a different function. S-allantoin, a major ureide compound, is produced in plant peroxisomes from oxidized purines. Sequence evidence suggested that the Transthyretin-like (TTL) protein, which interacts with brassinosteroid receptors, may act as a bifunctional enzyme in the synthesis of S-allantoin. Here, we show that recombinant TTL from Arabidopsis thaliana catalyzes two enzymatic reactions leading to the stereoselective formation of S-allantoin, hydrolysis of hydroxyisourate through a C-terminal Urah domain, and decarboxylation of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline through an N-terminal Urad domain. We found that two different mRNAs are produced from the TTL gene through alternative use of two splice acceptor sites. The corresponding proteins differ in the presence (TTL1−) and the absence (TTL2−) of a rare internal peroxisomal targeting signal (PTS2). The two proteins have similar catalytic activity in vitro but different in vivo localization: TTL1− localizes in peroxisomes, whereas TTL2− localizes in the cytosol. Similar splice variants are present in monocots and dicots. TTL originated in green algae through a Urad-Urah fusion, which entrapped an N-terminal PTS2 between the two domains. The presence of this gene in all Viridiplantae indicates that S-allantoin biosynthesis has general significance in plant nitrogen metabolism, while conservation of alternative splicing suggests that this mechanism has general implications in the regulation of the ureide pathway in flowering plants.

Chemical basis of nitrogen recovery through the ureide pathway: formation and hydrolysis of S-ureidoglycine in plants and bacteria.

While some organisms, including humans, eliminate oxidized purines to get rid of excess nitrogen, for many others the recovery of the purine ring nitrogen is vital. In the so-called ureide pathway, nitrogen is released as ammonia from allantoate through a series of reactions starting with allantoate amidohydrolase (AAH), a manganese-dependent enzyme found in plants and bacteria. We report NMR evidence that the true product of the AAH reaction is S-ureidoglycine, a nonstandard alpha-amino acid that spontaneously releases ammonia in vitro. Using gene proximity and logical genome analysis, we identified a candidate gene (ylbA) for S-ureidoglycine metabolism. The proteins encoded by Escherichia coli and Arabidopsis thaliana genes catalyze the manganese-dependent release of ammonia through hydrolysis of S-ureidoglycine. Hydrolysis then inverts the configuration and yields S-ureidoglycolate. S-Ureidoglycine aminohydrolase (UGHY) is cytosolic in bacteria, whereas in plants it is localized, like allantoate amidohydrolase, in the endoplasmic reticulum. These findings strengthen the basis for the known sensitivity of the ureide pathway to Mn availability and suggest a further rationale for the active transport of Mn in the endoplasmic reticulum of plant cells.

The Structure of 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase Provides Insights into the Mechanism of Uric Acid Degradation

The complete degradation of uric acid to (S)-allantoin, as recently elucidated, involves three enzymatic reactions. Inactivation by pseudogenization of the genes of the pathway occurred during hominoid evolution, resulting in a high concentration of urate in the blood and susceptibility to gout. Here, we describe the 1.8Å resolution crystal structure of the homodimeric 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, which catalyzes the last step in the urate degradation pathway, for both ligand-free enzyme and enzyme in complex with the substrate analogs (R)-allantoin and guanine. Each monomer comprises ten α-helices, grouped into two domains and assembled in a novel fold. The structure and the mutational analysis of the active site have allowed us to identify some residues that are essential for catalysis, among which His-67 and Glu-87 appear to play a particularly significant role. Glu-87 may facilitate the exit of the carboxylate group because of electrostatic repulsion that destabilizes the ground state of the substrate, whereas His-67 is likely to be involved in a protonation step leading to the stereoselective formation of the (S)-allantoin enantiomer as reaction product. The structural and functional characterization of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase can provide useful information in view of the potential use of this enzyme in the enzymatic therapy of gout.

Logical Identification of an Allantoinase Analog (puuE) Recruited from Polysaccharide Deacetylases

The hydrolytic cleavage of the hydantoin ring of allantoin, catalyzed by allantoinase, is required for the utilization of the nitrogen present in purine-derived compounds. The allantoinase gene (DAL1), however, is missing in many completely sequenced organisms able to use allantoin as a nitrogen source. Here we show that an alternative allantoinase gene (puuE) can be precisely identified by analyzing its logic relationship with three other genes of the pathway. The novel allantoinase is annotated in structure and sequence data bases as polysaccharide deacetylase for its homology with enzymes that catalyze hydrolytic reactions on chitin or peptidoglycan substrates. The recombinant PuuE protein from Pseudomonas fluorescens exhibits metal-independent allantoinase activity and stereospecificity for the S enantiomer of allantoin. The crystal structures of the protein and of protein-inhibitor complexes reveal an overall similarity with the polysaccharide deacetylase β/α barrel and remarkable differences in oligomeric assembly and active site geometry. The conserved Asp-His-His metal-binding triad is replaced by Glu-His-Trp, a configuration that is distinctive of PuuE proteins within the protein family. An extra domain at the top of the barrel offers a scaffold for protein tetramerization and forms a small substrate-binding cleft by hiding the large binding groove of polysaccharide deacetylases. Substrate positioning at the active site suggests an acid/base mechanism of catalysis in which only one member of the catalytic pair of polysaccharide deacetylases has been conserved. These data provide a structural rationale for the shifting of substrate specificity that occurred during evolution.

Nucleosome Depletion Activates Poised RNA Polymerase III at Unconventional Transcription Sites in Saccharomyces cerevisiae

RNA polymerase (pol) III, assisted by the transcription factors TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete pol III transcription complexes (Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118-4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1- and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNAIle ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c open reading frame, was found to be the strictly conserved remnant of an ancient tDNAArg. The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.