Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Noncanonical prokaryotic X family DNA polymerases lack polymerase activity and act as exonucleases.

The X family polymerases (PolXs) are specialized DNA polymerases that are found in all domains of life. While the main representatives of eukaryotic PolXs, which have dedicated functions in DNA repair, were studied in much detail, the functions and diversity of prokaryotic PolXs have remained largely unexplored. Here, by combining a comprehensive bioinformatic analysis of prokaryotic PolXs and biochemical experiments involving selected recombinant enzymes, we reveal a previously unrecognized group of PolXs that seem to be lacking DNA polymerase activity. The noncanonical PolXs contain substitutions of the key catalytic residues and deletions in their polymerase and dNTP binding sites in the palm and fingers domains, but contain functional nuclease domains, similar to canonical PolXs. We demonstrate that representative noncanonical PolXs from the Deinococcus genus are indeed inactive as DNA polymerases but are highly efficient as 3'-5' exonucleases. We show that both canonical and noncanonical PolXs are often encoded together with the components of the non-homologous end joining pathway and may therefore participate in double-strand break repair, suggesting an evolutionary conservation of this PolX function. This is a remarkable example of polymerases that have lost their main polymerase activity, but retain accessory functions in DNA processing and repair.

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure.

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB).

BACKGROUND: The rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P's sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date. RESULTS: Of the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation. CONCLUSIONS: The coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

The complex phylogenetic relationships of a 4mC/6mA DNA methyltransferase in prokaryotes.

DNA methyltransferases are proteins that modify DNA via attachment of methyl groups to nucleobases and are ubiquitous across the bacterial, archaeal, and eukaryotic domains of life. Here, we investigated the complex evolutionary history of the large and consequential 4mC/6mA DNA methyltransferase protein family using phylogenetic reconstruction of amino acid sequences. We present a well-supported phylogeny of this family based on systematic sampling of taxa across superphyla of bacteria and archaea. We compared the phylogeny to a current representation of the species tree of life and found that the 4mC/6mA methyltransferase family has a strikingly complex evolutionary history that likely began sometime after the last universal common ancestor of life diverged into the bacterial and archaeal lineages and probably involved many horizontal gene transfers within and between domains. Despite the complexity of its evolutionary history, we inferred that only one significant shift in molecular evolutionary rate characterizes the diversification of this protein family.

Structural variations of photosystem I-antenna supercomplex in response to adaptations to different light environments.

Photosystem I (PSI) is one of the two photosystems in photosynthesis, and generates reducing power required for carbon dioxide fixation. PSI exists as a reaction center core in cyanobacteria but is surrounded by light-harvesting antenna complexes (LHCI) to form PSI-LHCI supercomplexes in eukaryotic organisms. The structures of PSI core and PSI-LHCI have been reported from various organisms. We compare these structures and highlight the differences among different organisms. While the PSI core is more conserved, there are differences in its subunit composition and organization. Larger differences are found in the subunit composition, organization, and pigment binding in LHCI. All these changes can be explained in the framework of better adaptation to different light environment that each photosynthetic organism inhabits.

Biosynthesis and characterization of a recombinant eukaryotic allophycocyanin using prokaryotic accessory enzymes.

Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αß subunits that oligomerizes as a trimer (αß)3 . Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and ß subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λmaxAbs , λmaxEm , fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high-quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes.

Characterization and engineering of a DNA polymerase reveals a single amino-acid substitution in the fingers subdomain to increase strand-displacement activity of A-family prokaryotic DNA polymerases.

BACKGROUND: The discovery of thermostable DNA polymerases such as Taq DNA polymerase revolutionized amplification of DNA by polymerase chain reaction methods that rely on thermal cycling for strand separation. These methods are widely used in the laboratory for medical research, clinical diagnostics, criminal forensics and general molecular biology research. Today there is a growing demand for on-site molecular diagnostics; so-called 'Point-of-Care tests'. Isothermal nucleic acid amplification techniques do not require a thermal cycler making these techniques more suitable for performing Point-of-Care tests at ambient temperatures compared to traditional polymerase chain reaction methods. Strand-displacement activity is essential for such isothermal nucleic acid amplification; however, the selection of DNA polymerases with inherent strand-displacement activity that are capable of performing DNA synthesis at ambient temperatures is currently limited. RESULTS: We have characterized the large fragment of a DNA polymerase I originating from the marine psychrophilic bacterium Psychrobacillus sp. The enzyme showed optimal polymerase activity at pH 8-9 and 25-110 mM NaCl/KCl. The polymerase was capable of performing polymerase as well as robust strand-displacement DNA synthesis at ambient temperatures (25-37 °C). Through molecular evolution and screening of thousand variants we have identified a single amino-acid exchange of Asp to Ala at position 422 which induced a 2.5-fold increase in strand-displacement activity of the enzyme. Transferring the mutation of the conserved Asp residue to corresponding thermophilic homologues from Ureibacillus thermosphaericus and Geobacillus stearothermophilus also resulted in a significant increase in the strand-displacement activity of the enzymes. CONCLUSIONS: Substituting Asp with Ala at positon 422 resulted in a significant increase in strand-displacement activity of three prokaryotic A-family DNA polymerases adapted to different environmental temperatures i.e. being psychrophilic and thermophilic of origin. This strongly indicates an important role for the 422 position and the O1-helix for strand-displacement activity of DNA polymerase I. The D422A variants generated here may be highly useful for isothermal nucleic acid amplification at a wide temperature scale.

DEAD-box ATPases are global regulators of phase-separated organelles.

The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules1,2. How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs)3 are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles.

Evolution of protein kinase substrate recognition at the active site.

Protein kinases catalyse the phosphorylation of target proteins, controlling most cellular processes. The specificity of serine/threonine kinases is partly determined by interactions with a few residues near the phospho-acceptor residue, forming the so-called kinase-substrate motif. Kinases have been extensively duplicated throughout evolution, but little is known about when in time new target motifs have arisen. Here, we show that sequence variation occurring early in the evolution of kinases is dominated by changes in specificity-determining residues. We then analysed kinase specificity models, based on known target sites, observing that specificity has remained mostly unchanged for recent kinase duplications. Finally, analysis of phosphorylation data from a taxonomically broad set of 48 eukaryotic species indicates that most phosphorylation motifs are broadly distributed in eukaryotes but are not present in prokaryotes. Overall, our results suggest that the set of eukaryotes kinase motifs present today was acquired around the time of the eukaryotic last common ancestor and that early expansions of the protein kinase fold rapidly explored the space of possible target motifs.

ProGlycProt V2.0, a repository of experimentally validated glycoproteins and protein glycosyltransferases of prokaryotes.

Knowledge of glycosylation status and glycan-pattern of proteins are of considerable medical, academic and application interest. ProGlycProt V2.0 (www.proglycprot.org) therefore, is conceived and maintained as an exclusive web-resource providing comprehensive information on experimentally validated glycoproteins and protein glycosyltransferases (GTs) of prokaryotic origin. The second release of ProGlycProt features a major update with a 191% increase in the total number of entries, manually collected and curated from 607 peer-reviewed publications, on the subject. Protein GTs from prokaryotes that catalyze a varied range of glycan linkages are amenable glycoengineering tools. Therefore, the second release presents content that is greatly expanded and reorganized in two sub-databases: ProGPdb and ProGTdb. While ProGPdb provides information about validated glycoproteins (222 entries), ProGTdb catalogs enzymes/proteins that are instrumental in protein glycosylation, directly (122) or as accessory proteins (182). ProGlycProt V2.0 remains highly cross-referenced yet exclusive and complementary in content to other related databases. The second release further features enhanced search capability, a "compare" entries option and an innovative geoanalytical tool (MapView) facilitating location-assisted search-cum filtering of the entries using geo-positioning information of researchers/groups cited in the ProGlycProt V2.0 databases. Thus, ProGlycProt V2.0 continues to serve as a useful one-point web-resource on various evidence-based information on protein glycosylation in prokaryotes.

Two aspartate residues close to the lesion binding site of Agrobacterium (6-4) photolyase are required for Mg2+ stimulation of DNA repair.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg2+ . We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA-binding site are essential for the effect of Mg2+ . Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+ . In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron-sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron-sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment.

Ancient Transcription Factors in the News.

In every cell from bacteria to mammals, NusG-like proteins bind transcribing RNA polymerase to modulate the rate of nascent RNA synthesis and to coordinate it with numerous cotranscriptional processes that ultimately determine the transcript fate. Housekeeping NusG factors regulate expression of the bulk of the genome, whereas their highly specialized paralogs control just a few targets. In Escherichiacoli, NusG stimulates silencing of horizontally acquired genes, while its paralog RfaH counters NusG action by activating a subset of these genes. Acting alone or as part of regulatory complexes, NusG factors can promote uninterrupted RNA synthesis, bring about transcription pausing or premature termination, modulate RNA processing, and facilitate translation. Recent structural and mechanistic studies of NusG homologs from all domains of life reveal molecular details of multifaceted interactions that underpin their unexpectedly diverse regulatory roles. NusG proteins share conserved binding sites on RNA polymerase and many effects on the transcription elongation complex but differ in their mechanisms of recruitment, interactions with nucleic acids and secondary partners, and regulatory outcomes. Strikingly, some can alternate between autoinhibited and activated states that possess dramatically different secondary structures to achieve exquisite target specificity.

Evolutionary and structure-function analysis elucidates diversification of prokaryotic and eukaryotic trehalases.

Trehalase catalyses the breakdown of trehalose into two glucose moieties and is ubiquitous in all organisms. Here, we provide insights into the enigmatic origin and evolution of trehalase in major species. Study of taxonomic distribution, orthology, phylogeny and functional domains indicated that trehalase possibly originates from bacteria and was transmitted to other taxa through horizontal gene transfer. Domain analysis showed that glycosyl hydrolase family 37 is present in most of the sequences and represents dominant activity during evolution, and also, illustrating that cytosolic trehalase is primitive than its transmembrane form. Furthermore, it was observed that trehalase went through domain rearrangement to facilitate its activity in adverse environmental conditions like acidic pH. Gene context analysis depicts that trehalase neighbourhood consists of sugar transport and lipid metabolism genes. This highlights their relatedness in metabolic activity and similarity in gene regulation, respectively. Evolutionary and selection pressure analysis demonstrated that trehalase genes were duplicated and evolved under purifying selection, following horizontal gene transfer. Moreover, site-specific rate of evolution emphasized conservation of functionally important residues. In comparison with acid trehalase, neutral trehalase has an extra N-terminal extension. This study serves as an instigation to understand evolution and functionality of trehalase across diverse species. Communicated by Ramaswamy H. Sarma.

Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.

Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.

Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases.

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.

Role of DHH superfamily proteins in nucleic acids metabolism and stress tolerance in prokaryotes and eukaryotes.

DHH superfamily proteins play pivotal roles in various cellular processes like replication, recombination, repair and nucleic acids metabolism. These proteins are important for homeostasis maintenance and stress tolerance in prokaryotes and eukaryotes. The prominent members of DHH superfamily include single-strand specific exonuclease RecJ, nanoRNases, polyphosphatase PPX1, pyrophosphatase, prune phosphodiesterase and cell cycle protein Cdc45. The mutations of genes coding for DHH superfamily proteins lead to severe growth defects and in some cases, may be lethal. The members of superfamily have a wide substrate spectrum. The spectrum of substrate for DHH superfamily members ranges from smaller molecules like pyrophosphate and cyclic nucleotides to longer single-stranded DNA molecule. Several genetic, structural and biochemical studies have provided interesting insights about roles of DHH superfamily members. However, there are still various unexplored members in both prokaryotes and eukaryotes. Many aspects of this superfamily associated with homeostasis maintenance and stress tolerance are still not clearly understood. A comprehensive understanding is pre-requisite to decipher the physiological significance of members of DHH superfamily. This article provides the current understanding of DHH superfamily members and their significance in nucleic acids metabolism and stress tolerance across diverse forms of life.

Dual-Family Peptidylprolyl Isomerases (Immunophilins) of Select Monocellular Organisms.

The dual-family peptidylprolyl cis-trans isomerases (immunophilins) represent a naturally occurring chimera of the classical FK506-binding protein (FKBP) and cyclophilin (CYN), connected by a flexible linker. They are found exclusively in monocellular organisms. The modular builds of these molecules represent two distinct types: CYN-(linker)-FKBP and FKBP-3TPR (tetratricopeptide repeat)-CYN. Abbreviated respectively as CFBP and FCBP, the two classes also exhibit distinct organism preference, the CFBP being found in prokaryotes, and the FCBP in eukaryotes. This review summarizes the mystery of these unique class of prolyl isomerases, focusing on their host organisms, potential physiological role, and likely routes of evolution.

Application of eukaryotic and prokaryotic laccases in biosensor and biofuel cells: recent advances and electrochemical aspects.

Laccases exhibit a wide range of applications, especially in the electrochemical field, where they are regarded as a potential biotic component. Laccase-based biosensors have immense practical applications in the food, environmental, and medical fields. The application of laccases as biocathodes in enzymatic biofuel cells has promising potential in the preparation of implantable equipment. Extensive studies have been directed towards the potential role of fungal laccases as biotic components of electrochemical equipment. In contrast, the potential of prokaryotic laccases in electrochemistry has been not fully understood. However, there has been recent and rapid progress in the discovery and characterization of new types of prokaryotic laccases. In this review, we have comprehensively discussed the application of different sources of laccases as a biocatalytic component in various fields of application. Further, we described the potential of different types of laccases in bioelectrochemical applications.

Future perception in P450 research.

Cytochrome P450 (P450) of eukaryotes and prokaryotes diversified remarkably during the long course of their evolution. Since the diversification of P450 was the consequence of the adaptation of various living organisms to diverse environmental changes, the catalytic activities and physiological functions of the P450s of different taxa can be significantly different. It is likely that many P450s with novel physiological functions will be found in future when we further identify and examine the P450s of various eukaryotes and prokaryotes. The whole-genome sequences of a few thousand species are now available. The genome data will be helpful for the search of novel P450s in the eukaryotic and prokaryotic organisms that have not been studied so far. Discovery of new P450s with unique catalytic activities will expand the scope of biochemical and biophysical research on P450.