The Apo-structure of the Low Molecular Weight Protein-tyrosine Phosphatase A (MptpA) from Mycobacterium tuberculosis Allows for Better Target-specific Drug Development

Protein-tyrosine phosphatases (PTPs) and protein-tyrosine kinases co-regulate cellular processes. In pathogenic bacteria, they are frequently exploited to act as key virulence factors for human diseases. Mycobacterium tuberculosis, the causative organism of tuberculosis, secretes a low molecular weight PTP (LMW-PTP), MptpA, which is required for its survival upon infection of host macrophages. Although there is otherwise no sequence similarity of LMW-PTPs to other classes of PTPs, the phosphate binding loop (P-loop) CX₅R and the loop containing a critical aspartic acid residue (D-loop), required for the catalytic activity, are well conserved. In most high molecular weight PTPs, ligand binding to the P-loop triggers a large conformational reorientation of the D-loop, in which it moves ∼10 Å, from an “open” to a “closed” conformation. Until now, there have been no ligand-free structures of LMW-PTPs described, and hence the dynamics of the D-loop have remained largely unknown for these PTPs. Here, we present a high resolution solution NMR structure of the free form of the MptpA LMW-PTP. In the absence of ligand and phosphate ions, the D-loop adopts an open conformation. Furthermore, we characterized the binding site of phosphate, a competitive inhibitor of LMW-PTPs, on MptpA and elucidated the involvement of both the P- and D-loop in phosphate binding. Notably, in LMW-PTPs, the phosphorylation status of two well conserved tyrosine residues, typically located in the D-loop, regulates the enzyme activity. PtkA, the kinase complementary to MptpA, phosphorylates these two tyrosine residues in MptpA. We characterized the MptpA-PtkA interaction by NMR spectroscopy to show that both the P- and D-loop form part of the binding interface.     

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins (“tāpirins,” origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.     

Unexpected Population Distribution in a Microbial Mat Community: Sulfate-Reducing Bacteria Localized to the Highly Oxic Chemocline in Contrast to a Eukaryotic Preference for Anoxia

The distribution and abundance of sulfate-reducing bacteria (SRB) and eukaryotes within the upper 4 mm of a hypersaline cyanobacterial mat community were characterized at high resolution with group-specific hybridization probes to quantify 16S rRNA extracted from 100-μm depth intervals. This revealed a preferential localization of SRB within the region defined by the oxygen chemocline. Among the different groups of SRB quantified, including members of the provisional families “Desulfovibrionaceae” and “Desulfobacteriaceae,” Desulfonema-like populations dominated and accounted for up to 30% of total rRNA extracted from certain depth intervals of the chemocline. These data suggest that recognized genera of SRB are not necessarily restricted by high levels of oxygen in this mat community and the possibility of significant sulfur cycling within the chemocline. In marked contrast, eukaryotic populations in this community demonstrated a preference for regions of anoxia.     

Conserved sequence motifs among bacterial,eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily.

Members of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol-phosphatase domains of the bacterial bifunctional enzymes imidazole-glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose-6-phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]-D-[ILFRMVY]-D-[GSNDE]-[TV]-[ILVAM]-[AT S VILMC]-X-¿YFWHKR)-X-¿YFWHNQ¿-X( 102,191)-¿KRHNQ¿-G-D-¿FYWHILVMC¿-¿QNH¿-¿FWYGP¿-D -¿PSNQYW¿. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as "DDDD" after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.     

"PP2C7s", Genes Most Highly Elaborated in Photosynthetic Organisms,Reveal the Bacterial Origin and Stepwise Evolution of PPM/PP2C Protein Phosphatases

Mg⁺²/Mn⁺²-dependent type 2C protein phosphatases (PP2Cs) are ubiquitous in eukaryotes, mediating diverse cellular signaling processes through metal ion catalyzed dephosphorylation of target proteins. We have identified a distinct PP2C sequence class (“PP2C7s”) which is nearly universally distributed in Eukaryotes, and therefore apparently ancient. PP2C7s are by far most prominent and diverse in plants and green algae. Combining phylogenetic analysis, subcellular localization predictions, and a distillation of publically available gene expression data, we have traced the evolutionary trajectory of this gene family in photosynthetic eukaryotes, demonstrating two major sequence assemblages featuring a succession of increasingly derived sub-clades. These display predominant expression moving from an ancestral pattern in photosynthetic tissues toward non-photosynthetic, specialized and reproductive structures. Gene co-expression network composition strongly suggests a shifting pattern of PP2C7 gene functions, including possible regulation of starch metabolism for one homologue set in Arabidopsis and rice. Distinct plant PP2C7 sub-clades demonstrate novel amino terminal protein sequences upon motif analysis, consistent with a shifting pattern of regulation of protein function. More broadly, neither the major events in PP2C sequence evolution, nor the origin of the diversity of metal binding characteristics currently observed in different PP2C lineages, are clearly understood. Identification of the PP2C7 sequence clade has allowed us to provide a better understanding of both of these issues. Phylogenetic analysis and sequence comparisons using Hidden Markov Models strongly suggest that PP2Cs originated in Bacteria (Group II PP2C sequences), entered Eukaryotes through the ancestral mitochondrial endosymbiosis, elaborated in Eukaryotes, then re-entered Bacteria through an inter-domain gene transfer, ultimately producing bacterial Group I PP2C sequences. A key evolutionary event, occurring first in ancient Eukaryotes, was the acquisition of a conserved aspartate in classic Motif 5. This has been inherited subsequently by PP2C7s, eukaryotic PP2Cs and bacterial Group I PP2Cs, where it is crucial to the formation of a third metal binding pocket, and catalysis.     

Powerful Sequence Similarity Search Methods and In-Depth Manual Analyses Can Identify Remote Homologs in Many Apparently “Orphan” Viral Proteins

The genome sequences of new viruses often contain many “orphan” or “taxon-specific” proteins apparently lacking homologs. However, because viral proteins evolve very fast, commonly used sequence similarity detection methods such as BLAST may overlook homologs. We analyzed a data set of proteins from RNA viruses characterized as “genus specific” by BLAST. More powerful methods developed recently, such as HHblits or HHpred (available through web-based, user-friendly interfaces), could detect distant homologs of a quarter of these proteins, suggesting that these methods should be used to annotate viral genomes. In-depth manual analyses of a subset of the remaining sequences, guided by contextual information such as taxonomy, gene order, or domain cooccurrence, identified distant homologs of another third. Thus, a combination of powerful automated methods and manual analyses can uncover distant homologs of many proteins thought to be orphans. We expect these methodological results to be also applicable to cellular organisms, since they generally evolve much more slowly than RNA viruses. As an application, we reanalyzed the genome of a bee pathogen, Chronic bee paralysis virus (CBPV). We could identify homologs of most of its proteins thought to be orphans; in each case, identifying homologs provided functional clues. We discovered that CBPV encodes a domain homologous to the Alphavirus methyltransferase-guanylyltransferase; a putative membrane protein, SP24, with homologs in unrelated insect viruses and insect-transmitted plant viruses having different morphologies (cileviruses, higreviruses, blunerviruses, negeviruses); and a putative virion glycoprotein, ORF2, also found in negeviruses. SP24 and ORF2 are probably major structural components of the virions.     

Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation.     

Three Prochlorococcus cyanophage genomes: signature features and ecological interpretations

The oceanic cyanobacteria Prochlorococcus are globally important, ecologically diverse primary producers. It is thought that their viruses (phages) mediate population sizes and affect the evolutionary trajectories of their hosts. Here we present an analysis of genomes from three Prochlorococcus phages: a podovirus and two myoviruses. The morphology, overall genome features, and gene content of these phages suggest that they are quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages. Using the existing phage taxonomic framework as a guideline, we examined genome sequences to establish “core” genes for each phage group. We found the podovirus contained 15 of 26 core T7-like genes and the two myoviruses contained 43 and 42 of 75 core T4-like genes. In addition to these core genes, each genome contains a significant number of “cyanobacterial” genes, i.e., genes with significant best BLAST hits to genes found in cyanobacteria. Some of these, we speculate, represent “signature” cyanophage genes. For example, all three phage genomes contain photosynthetic genes (psbA, hliP) that are thought to help maintain host photosynthetic activity during infection, as well as an aldolase family gene (talC) that could facilitate alternative routes of carbon metabolism during infection. The podovirus genome also contains an integrase gene (int) and other features that suggest it is capable of integrating into its host. If indeed it is, this would be unprecedented among cultured T7-like phages or marine cyanophages and would have significant evolutionary and ecological implications for phage and host. Further, both myoviruses contain phosphate-inducible genes (phoH and pstS) that are likely to be important for phage and host responses to phosphate stress, a commonly limiting nutrient in marine systems. Thus, these marine cyanophages appear to be variations of two well-known phages—T7 and T4—but contain genes that, if functional, reflect adaptations for infection of photosynthetic hosts in low-nutrient oceanic environments.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.     

Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions: DEVELOPMENT OF BIOENGINEERED PRENYLATION PATHWAYS*

Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

Human METTL20 Is a Mitochondrial Lysine Methyltransferase That Targets the β Subunit of Electron Transfer Flavoprotein (ETFβ) and Modulates Its Activity

Proteins are frequently modified by post-translational methylation of lysine residues, catalyzed by S-adenosylmethionine-dependent lysine methyltransferases (KMTs). Lysine methylation of histone proteins has been extensively studied, but it has recently become evident that methylation of non-histone proteins is also abundant and important. The human methyltransferase METTL20 belongs to a group of 10 established and putative human KMTs. We here found METTL20 to be associated with mitochondria and determined that recombinant METTL20 methylated a single protein in extracts from human cells. Using an methyltransferase activity-based purification scheme, we identified the β-subunit of the mitochondrially localized electron transfer flavoprotein (ETFβ) as the substrate of METTL20. Furthermore, METTL20 was found to specifically methylate two adjacent lysine residues, Lys²⁰⁰ and Lys²⁰³, in ETFβ both in vitro and in cells. Interestingly, the residues methylated by METTL20 partially overlap with the so-called “recognition loop” in ETFβ, which has been shown to mediate its interaction with various dehydrogenases. Accordingly, we found that METTL20-mediated methylation of ETFβ in vitro reduced its ability to receive electrons from the medium chain acyl-CoA dehydrogenase and the glutaryl-CoA dehydrogenase. In conclusion, the present study establishes METTL20 as the first human KMT localized to mitochondria and suggests that it may regulate cellular metabolism through modulating the interaction between its substrate ETFβ and dehydrogenases. Based on the previous naming of similar enzymes, we suggest the renaming of human METTL20 to ETFβ-KMT.     

Folding of a Cyclin Box: LINKING MULTITARGET BINDING TO MARGINAL STABILITY,OLIGOMERIZATION, AND AGGREGATION OF THE RETINOBLASTOMA TUMOR SUPPRESSOR AB POCKET DOMAIN*

The retinoblastoma tumor suppressor (Rb) controls the proliferation, differentiation, and survival of cells in most eukaryotes with a role in the fate of stem cells. Its inactivation by mutation or oncogenic viruses is required for cellular transformation and eventually carcinogenesis. The high conservation of the Rb cyclin fold prompted us to investigate the link between conformational stability and ligand binding properties of the RbAB pocket domain. RbAB unfolding presents a three-state transition involving cooperative secondary and tertiary structure changes and a partially folded intermediate that can oligomerize. The first transition corresponds to unfolding of the metastable B subdomain containing the binding site for the LXCXE motif present in cellular and viral targets, and the second transition corresponds to the stable A subdomain. The low thermodynamic stability of RbAB translates into a propensity to rapidly oligomerize and aggregate at 37 °C (T₅₀ = 28 min) that is suppressed by human papillomavirus E7 and E2F peptide ligands, suggesting that Rb is likely stabilized in vivo through binding to target proteins. We propose that marginal stability and associated oligomerization may be conserved for function as a “hub” protein, allowing the formation of multiprotein complexes, which could constitute a robust mechanism to retain its cell cycle regulatory role throughout evolution. Decreased stability and oligomerization are shared with the p53 tumor suppressor, suggesting a link between folding and function in these two essential cell regulators that are inactivated in most cancers and operate within multitarget signaling pathways.     

Mycorrhizal fungi induced activation of tomato defense system mitigates Fusarium wilt stress

The fungus Fusarium oxysporum f. sp. lycopersici (FOL) is known to cause vascular wilt on tomato almost over the world. Inoculation of FOL reduced plant growth and increased wilt of tomato. The following study examined the possible role of arbuscular mycorrhizal fungi (AMF) consortium comprising of Rhizophagus intraradices, Funneliformis mosseae and Claroideoglomus etunicatum against FOL in tomato and explored in an inducing plant systemic defense. AMF inoculation reduced the wilt disease within vascular tissue and in vivo production of fusaric acid was observed which may be responsible in reduced wilting. FOL had an antagonistic effect on AMF colonization, reduced the number of spores, arbuscules and vesicles. AMF also inhibited the damage induced by Fusarium wilt through increasing chlorophyll contents along with the activity of phosphate metabolising enzymes (acid and alkaline phosphatases). Moreover, tomato plants with mycorrhizal inoculation showed an increase in the level of antioxidant enzymes including glutathione reductase, catalase, and etc. with an ultimate influence on the elimination of reactive oxygen species. Moreover, rise in phosphatase along with antioxidant enzymatic systems and enhanced photosynthetic performance contributed to induced resistance against FOL in tomato.     

Evolution of Bacterial-Like Phosphoprotein Phosphatases in Photosynthetic Eukaryotes Features Ancestral Mitochondrial or Archaeal Origin and Possible Lateral Gene Transfer

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.Reversible protein phosphorylation is a posttranslational mechanism central to the proper function of living organisms (Brautigan, 2013). Governed by two large groups of enzymes, protein kinases and protein phosphatases, this mechanism has been suggested to regulate upwards of 70% of all eukaryotic proteins (Olsen et al., 2010). Protein phosphatases represent one-half of this dynamic regulatory system and have been shown to be highly regulated proteins themselves (Roy and Cyert, 2009; Shi, 2009; Uhrig et al., 2013). Classically, protein phosphatases have been placed into four families defined by a combination of their catalytic mechanisms, metal ion requirements, and phosphorylated amino acid targets (Kerk et al., 2008). These four families are the phosphoprotein phosphatases (PPPs), metallo-dependent protein phosphatases, protein Tyr phosphatases, and Asp-based phosphatases. The PPP protein phosphatases, best known to include PP1, PP2A, PP2B, and PP4 to PP7 (Kerk et al., 2008; Shi, 2009), have been found to regulate a diverse number of biological processes in plants ranging from cell signaling (Ahn et al., 2011; Di Rubbo et al., 2011; Tran et al., 2012) to metabolism (Heidari et al., 2011; Leivar et al., 2011) and hormone biosynthesis (Skottke et al., 2011). The classical PPP protein phosphatase family has been expanded to include three novel classes that show greatest similarity to PPP-like protein phosphatases of prokaryotic origin (Andreeva and Kutuzov, 2004; Uhrig and Moorhead, 2011a; Uhrig et al., 2013). These bacterial-like phosphatase classes were annotated as Shewanella-like (SLP) phosphatases, Rhizobiales-like (RLPH) phosphatases, and ApaH-like (ALPH) phosphatases based on their similarity to prokaryotic sequences from these respective sources (Andreeva and Kutuzov, 2004). Recent characterization of the SLP phosphatases from Arabidopsis (Arabidopsis thaliana) provided biochemical evidence of insensitivity to the classic PPP protein phosphatase inhibitors okadaic acid and microcystin in addition to revealing a lack of genetic redundancy across sequenced plant genomes (Uhrig and Moorhead, 2011a).The characterization of eukaryotic protein evolution can provide insight into individual protein or protein class conservation across the domains of life for biotechnological applications in addition to furthering our understanding of how multicellular life evolved. In particular, investigation into the evolution of key signaling proteins, such as protein kinases and phosphatases from plants, can have wide-ranging agribiotechnological and medical potential. This can include the development of healthier, disease- or stress-resistant crops in addition to treatments for parasitic organisms such as Plasmodium spp. (malaria; Patzewitz et al., 2013) and other chromoalveolates (Kutuzov and Andreeva, 2008; Uhrig and Moorhead, 2011b) that are derived from photosynthetic eukaryotes and maintain a remnant chloroplast (apicoplast; Le Corguillé et al., 2009; Janouskovec et al., 2010; Kalanon and McFadden, 2010; Walker et al., 2011). The existence of proteins that are conserved across diverse eukaryotic phyla but absent in metazoa, such as the majority of bacterial-like PPP protein phosphatases described here, presents unique research opportunities.Conventional understanding of the acquisition by eukaryotes of prokaryotic genes and proteins largely involves ancient endosymbiotic gene transfer events stemming from primary endosymbiosis of α-Proteobacteria and Cyanobacteria to form eukaryotic mitochondria and chloroplasts, respectively (Keeling and Palmer, 2008; Dorrell and Smith, 2011; Tirichine and Bowler, 2011). Over time, however, it has become apparent that alternative modes of eukaryotic gene and protein acquisition exist, such as independent horizontal or lateral gene transfer (LGT) events (Keeling and Palmer, 2008; Keeling, 2009). Targeted studies of protein evolution have seen a steady rise in documented LGT events across a wide variety of eukaryotic organisms, including photosynthetic eukaryotes (Derelle et al., 2006; Raymond and Kim, 2012; Schönknecht et al., 2013), nematodes (Mayer et al., 2011), arthropods (Acuña et al., 2012), fungi (Wenzl et al., 2005), amoebozoa (Clarke et al., 2013), and oomycetes (Belbahri et al., 2008). Each instance documents the integration of a bacterial gene(s) into a eukaryotic organism, seemingly resulting in an adaptive advantage(s) important to organism survival.Utilizing a number of in silico bioinformatic techniques and available sequenced genomes, the molecular evolution of three bacterial-like PPP classes found in eukaryotes is revealed to involve ancient mitochondrial or archaeal origin plus additional possible LGT events. A third, more ancient group of SLP phosphatases (SLP3 phosphatases) is defined in green algae. Subcellular localization predictions reveal distinctive subsets of bacterial-like PPPs, which may correlate with altered functions. In addition, the large sequence collections compiled here have allowed the elucidation of two highly conserved C-terminal domain motifs, which are specific to each bacterial-like PPP class and whose differences are particularly pronounced in photosynthetic eukaryotes. Together, these findings substantially expand our knowledge of the molecular evolution of the bacterial-like PPPs and point the way toward attractive future research avenues.     

The Crystal Structure of Arabidopsis VSP1 Reveals the Plant Class C-Like Phosphatase Structure of the DDDD Superfamily of Phosphohydrolases

Arabidopsis thaliana vegetative storage proteins, VSP1 and VSP2, are acid phosphatases and belong to the haloacid dehalogenase (HAD) superfamily. In addition to their potential nutrient storage function, they were thought to be involved in plant defense and flower development. To gain insights into the architecture of the protein and obtain clues about its function, we have tested their substrate specificity and solved the structure of VSP1. The acid phosphatase activities of these two enzymes require divalent metal such as magnesium ion. Conversely, the activity of these two enzymes is inhibited by vanadate and molybdate, but is resistant to inorganic phosphate. Both VSP1 and VSP2 did not exhibit remarkable activities to any physiological substrates tested. In the current study, we presented the crystal structure of recombinant VSP1 at 1.8 Å resolution via the selenomethionine single-wavelength anomalous diffraction (SAD). Specifically, an α-helical cap domain on the top of the α/β core domain is found to be involved in dimerization. In addition, despite of the low sequence similarity between VSP1 and other HAD enzymes, the core domain of VSP1 containing conserved active site and catalytic machinery displays a classic haloacid dehalogenase fold. Furthermore, we found that VSP1 is distinguished from bacterial class C acid phosphatase P4 by several structural features. To our knowledge, this is the first study to reveal the crystal structure of plant vegetative storage proteins.     

O-GlcNAcase: Promiscuous Hexosaminidase or Key Regulator of O-GlcNAc Signaling?

O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches. This minireview charts the discovery and functional and structural analysis of OGA and summarizes the insights gained from recent studies using OGA inhibition, gene knock-out, and overexpression. We identify several areas of “known unknowns” that would benefit from future research, such as the enigmatic C-terminal domain of OGA.     

Microbial Protein-tyrosine Kinases

Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins.     

Molecular Characterization of “Candidatus Similichlamydia latridicola” gen. nov., sp. nov. (Chlamydiales: “Candidatus Parilichlamydiaceae”), a Novel Chlamydia-Like Epitheliocystis Agent in the Striped Trumpeter,Latris lineata (Forster)

Histological analysis of gill samples taken from individuals of Latris lineata reared in aquaculture in Tasmania, Australia, and those sampled from the wild revealed the presence of epitheliocystis-like basophilic inclusions. Subsequent morphological, in situ hybridization, and molecular analyses were performed to confirm the presence of this disease and discovered a Chlamydia-like organism associated with this condition, and the criteria set by Fredericks and Relman''s postulates were used to establish disease causation. Three distinct 16S rRNA genotypes were sequenced from 16 fish, and phylogenetic analyses of the nearly full-length 16S rRNA sequences generated for this bacterial agent indicated that they were nearly identical novel members of the order Chlamydiales. This new taxon formed a well-supported clade with “Candidatus Parilichlamydia carangidicola” from the yellowtail kingfish (Seriola lalandi). On the basis of sequence divergence over the 16S rRNA region relative to all other members of the order Chlamydiales, a new genus and species are proposed here for the Chlamydia-like bacterium from L. lineata, i.e., “Candidatus Similichlamydia latridicola” gen. nov., sp. nov.