Thiol dioxygenases: unique families of cupin proteins

Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.     

Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution

Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.     

Germin and Germin-like Proteins: Evolution,Structure, and Function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide–generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.     

Evolution of functional diversity in the cupin superfamily

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.     

Structure of a cupin protein Plu4264 from Photorhabdus luminescens subsp. laumondii TTO1 at 1.35 Å resolution

Proteins belonging to the cupin superfamily have a wide range of catalytic and noncatalytic functions. Cupin proteins commonly have the capacity to bind a metal ion with the metal frequently determining the function of the protein. We have been investigating the function of homologous cupin proteins that are conserved in more than 40 species of bacteria. To gain insights into the potential function of these proteins we have solved the structure of Plu4264 from Photorhabdus luminescens TTO1 at a resolution of 1.35 Å and identified manganese as the likely natural metal ligand of the protein. Proteins 2015; 83:383–388. © 2014 Wiley Periodicals, Inc.     

A multifunctional bicupin serves as precursor for a chromosomal protein of Pisum sativum seeds

The fact that the psp54 gene codes for p16, a seed chromatin protein of Pisum sativum, has been described previously. In the present paper it is shown that p54, the p16 precursor, also exists as a free polypeptide in pea and that it also yields p38, a second polypeptide from the N-terminal region of p54, which is co-localized at a subcellular level with p16. By using antibodies against pea p16 and p38, it was found that these proteins are present in the members of the tribe Viciae examined. Sequence analysis and 3D modelling indicates that p54 proteins belong to the cupin superfamily, and that they are related to sucrose binding proteins and, to a lesser extent, to vicilin-type seed storage proteins. Nevertheless, several distinctive characteristics of psp54 expression have been found: (i) the gene is differentially induced by ABA and several stress situations, in accordance with the presence of putative separate ABA and stress responsive elements in its promoter; (ii) the proteins are present in pods and seed coats, tissues of maternal origin; and (iii) p54 mRNA accumulates in the dry seeds. In view of both the functional properties of p54-derived proteins and the features of the psp54 gene expression, it is concluded that p54 represents a novel class within the cupin superfamily.     

Functional characterization of seed coat-specific members of the barley germin gene family

The present project aimed to isolate testa-, pericarp- and epicarp-specific gene promoters for the developing caryopsis of barley (Hordeum vulgare L.). These might be applied in transgenic plants to express antifungal agents or modify metabolic pathways. A testa-specific 379-nucleotide fragment was cloned by differential amplification and used to screen a bacterial artificial chromosome (BAC) library of 6.3 haploid genome equivalents. Fifty-three clones containing genes encoding for proteins of the germin family were found. Characterization of the clones identified a minimum of six seed coat- and eight leaf-specific germin genes. Four seed coat- and one leaf-specific genes were sequenced. The deduced primary structure of the proteins revealed a remarkable conservation of the manganese(II) binding His and Glu residues and β-barrel secondary structure of oxalate oxidase – also in barley, wheat, rice and Arabidopsis germins, for which an enzymatic activity has not yet been identified. The oxalate oxidase and germins of barley and other species are synthesized with a conserved pre-sequence of 23 or 24 amino acids for targeting into the cell wall. β-Glucuronidase expression with the barley germin F gene promoter occurs specifically in the testa and epicarp of the developing barley caryopsis, while expression with the B gene promoter is restricted to the testa. Oxalate oxidase activity is prominent in the epicarp and the root tips of the developing embryo. A family tree based on primary structure homologies of germins distinguishes three groups: oxalate oxidases, leaf-specific germins and seed coat-specific germins.     

Peptidoglycan recognition proteins of the innate immune system

Peptidoglycan (PGN) is the major component of bacterial cell walls and one of the main microbial products recognized by the innate immune system. PGN recognition is mediated by several families of pattern recognition molecules, including Toll-like receptors, nucleotide-binding oligomerization domain-containing proteins, and peptidoglycan recognition proteins (PGRPs). However, only the interaction of PGN with PGRPs, which are highly conserved from insects to mammals, has so far been characterized at the molecular level. Here, we describe recent structural studies of PGRPs that reveal the basis for PGN recognition and provide insights into the signal transduction and antibacterial activities of these innate immune proteins.     

The microbial and danger signals that activate Nod-like receptors

Nod-like receptors (NLRs) are a family of intracellular sensors that play key roles in innate immunity and inflammation. While some NLRs, including Nod1, Nod2, NAIP and IPAF, detect conserved bacterial molecular signatures from within the host cytosol, other members of this family seem to have evolved the capacity to sense danger signals perhaps independently of a microbial trigger. This is illustrated by the discovery that Nalp3 and Nalp1 are specifically activated by low concentrations of intracellular potassium. The fact that several stimuli, including bacterial toxins and some viruses, but also sterile crystals made of uric acid, asbestos or aluminium hydroxide, can trigger the Nalp3 inflammasome illustrate the fascinating prospect that microbial infections and certain danger signals may be perceived similarly by host recognition systems. Gaining insight into the function of NLR proteins in general will impact considerably on our understanding of the mechanisms underlying immunity to infection, adjuvanticity and auto-inflammatory disorders. In this review, we summarize the current knowledge on the microbial- and danger-derived signals that activate NLRs.     

A novel class of modular transporters for vitamins in prokaryotes

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters.     

JmjC: cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta

On the basis of significant sequence similarity, we have identified JmjC domains in more than 100 eukaryotic and bacterial sequences. These include human hairless, mutated in individuals with alopecia universalis, retinoblastoma-binding protein 2 and several putative chromatin-associated proteins. JmjC domains are predicted to be metalloenzymes that adopt the cupin fold, and are candidates for enzymes that regulate chromatin remodelling.     

The gamma-core motif correlates with antimicrobial activity in cysteine-containing kaliocin-1 originating from transferrins

Kaliocin-1 is a 31-residue peptide derived from human lactoferrin, and with antimicrobial properties that recapitulate those of its 611 amino acid parent holoprotein. As kaliocin-1 is a cysteine-stabilized peptide, it was of interest to determine whether it contained a multidimensional gamma-core signature recently identified as common to virtually all classes of disulfide-stabilized antimicrobial peptides. Importantly, sequence and structural analyses identified an iteration of this multidimensional antimicrobial signature in kaliocin-1. Further, the gamma-core motif was found to be highly conserved in the transferrin family of proteins across the phylogenetic spectrum. Previous studies suggested that the mechanism by which kaliocin-1 exerts anti-candidal efficacy depends on mitochondrial perturbation without cell membrane permeabilization. Interestingly, results of a yeast two-hybrid screening analysis identified an interaction between kaliocin-1 and mitochondrial initiation factor 2 in a Saccharomyces cerevisiae model system. Taken together, these data extend the repertoire of antimicrobial peptides that contain gamma-core motifs, and suggest that the motif is conserved within large native as well as antimicrobial peptide subcomponents of transferrin family proteins. Finally, these results substantiate the hypothesis that antimicrobial activity associated with host defense effector proteins containing a gamma-core motif may correspond to targets common to fungal mitochondria or their bacterial ancestors.     

Exploiting genomic patterns to discover new supramolecular protein assemblies

Bacterial microcompartments are supramolecular protein assemblies that function as bacterial organelles by compartmentalizing particular enzymes and metabolic intermediates. The outer shells of these microcompartments are assembled from multiple paralogous structural proteins. Because the paralogs are required to assemble together, their genes are often transcribed together from the same operon, giving rise to a distinctive genomic pattern: multiple, typically small, paralogous proteins encoded in close proximity on the bacterial chromosome. To investigate the generality of this pattern in supramolecular assemblies, we employed a comparative genomics approach to search for protein families that show the same kind of genomic pattern as that exhibited by bacterial microcompartments. The results indicate that a variety of large supramolecular assemblies fit the pattern, including bacterial gas vesicles, bacterial pili, and small heat‐shock protein complexes. The search also retrieved several widely distributed protein families of presently unknown function. The proteins from one of these families were characterized experimentally and found to show a behavior indicative of supramolecular assembly. We conclude that cotranscribed paralogs are a common feature of diverse supramolecular assemblies, and a useful genomic signature for discovering new kinds of large protein assemblies from genomic data.     

MOSC domains: ancient, predicted sulfur-carrier domains, present in diverse metal-sulfur cluster biosynthesis proteins including Molybdenum cofactor sulfurases

Using computational analysis, a novel superfamily of beta-strand-rich domains was identified in the Molybdenum cofactor sulfurase and several other proteins from both prokaryotes and eukaryotes. These MOSC domains contain an absolutely conserved cysteine and occur either as stand-alone forms such as the bacterial YiiM proteins, or fused to other domains such as a NifS-like catalytic domain in Molybdenum cofactor sulfurase. The MOSC domain is predicted to be a sulfur-carrier domain that receives sulfur abstracted by the pyridoxal phosphate-dependent NifS-like enzymes, on its conserved cysteine, and delivers it for the formation of diverse sulfur-metal clusters. The identification of this domain may clarify the mechanism of biogenesis of various metallo-enzymes including Molybdenum cofactor-containing enzymes that are compromised in human type II xanthinuria.     

Globulins are the main seed storage proteins in Brachypodium distachyon

Brachypodium distachyon is being developed as a model system to study temperate cereals and forage grasses. We have begun to investigate its utility to understand seed development and grain filling by identifying the major seed storage proteins in a diploid accession Bd21. With the use of ID SDS-PAGE and mass spectrometry we detected seven major storage protein bands, six of which were identified as globulins. A subset of the major seed proteins isolated from three hexaploid accessions, Bd4, Bd14 and Bd17 were also identified as globulins. Several Brachypodium cDNAs clones encoding globulin were completely sequenced. Two types of globulin genes were identified, Bd.glo1 and Bd.glo2, which are similar to maize 7S and oat 12S globulins, respectively. The derived polypeptide sequences of the globulins contain a typical signal peptide sequence in their polypeptide N-termini and two cupin domains. Bd.glo1 is encoded by a single copy gene, whereas, Bd.glo2 belongs to a gene family.     

Crystal structure of Pyrococcus furiosus phosphoglucose isomerase. Implications for substrate binding and catalysis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between d-fructose 6-phosphate and d-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-A resolution. Structural comparison of this archaeal PGI with the previously solved structures of bacterial and eukaryotic PGIs reveals a completely different structure. Each subunit of the homodimeric Pfu PGI consists of a cupin domain, for which the overall structure is similar to other cupin domain-containing proteins, and includes a conserved transition metal-binding site. Biochemical data on the recombinant enzyme suggests that Fe2+ is bound to Pfu PGI. However, as catalytic activity is not strongly influenced either by the replacement of Fe2+ by a range of transition metals or by the presence or absence of the bound metal ion, we suggest that the metal may not be directly involved in catalysis but rather may be implicated in substrate recognition.