Bacteriophage therapy against pathological Klebsiella pneumoniae ameliorates the course of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by progressive biliary inflammation and fibrosis. Although gut commensals are associated with PSC, their causative roles and therapeutic strategies remain elusive. Here we detect abundant Klebsiella pneumoniae (Kp) and Enterococcus gallinarum in fecal samples from 45 PSC patients, regardless of intestinal complications. Carriers of both pathogens exhibit high disease activity and poor clinical outcomes. Colonization of PSC-derived Kp in specific pathogen-free (SPF) hepatobiliary injury-prone mice enhances hepatic Th17 cell responses and exacerbates liver injury through bacterial translocation to mesenteric lymph nodes. We developed a lytic phage cocktail that targets PSC-derived Kp with a sustained suppressive effect in vitro. Oral administration of the phage cocktail lowers Kp levels in Kp-colonized germ-free mice and SPF mice, without off-target dysbiosis. Furthermore, we demonstrate that oral and intravenous phage administration successfully suppresses Kp levels and attenuates liver inflammation and disease severity in hepatobiliary injury-prone SPF mice. These results collectively suggest that using a lytic phage cocktail shows promise for targeting Kp in PSC.

Characterization of ancestral Fe/Mn superoxide dismutases indicates their cambialistic origin.

Superoxide dismutases (SODs) are critical metalloenzymes mitigating the damages of the modern oxygenated world. However, the emergence of one family of SODs, the Fe/Mn SOD, has been recurrently proposed to predate the great oxygenation event (GOE). This ancient family lacks metal binding selectivity, but displays strong catalytic selectivity. Therefore, some homologues would only be active when bound to Fe or Mn, although others, dubbed cambialistic, would function when loaded with either ion. This posed the longstanding question about the identity of the cognate metal ion of the first SODs to emerge. In this work, we utilize ancestral sequence reconstruction techniques to infer the earliest SODs. We show that the "ancestors" are active in vivo and in vitro. Further, we test their metal specificity and demonstrate that they are cambialistic in nature. Our findings shed light on how the predicted Last Common Universal Ancestor was capable of dealing with decomposition of the superoxide anion, and the early relationship between life, oxygen, and metal ion availability.

Innovation and tinkering in the evolution of oxidases.

Although molecular oxygen is a relative newcomer to the biosphere, it has had a profound impact on metabolism. About 700 oxygen-dependent enzymatic reactions are known, the vast majority of which emerged only after the appearance of oxygen in the biosphere, circa 3 billion years ago. Oxygen was a major driving force for evolutionary innovation-~60% of all known oxygen-dependent enzyme families emerged as such; that is, the founding ancestor was an O2 -dependent enzyme. The other 40% seem to have diverged by tinkering from pre-existing proteins whose function was not related to oxygen. Here, we focus on the latter. We describe transitions from various enzyme classes, as well as from non-enzymatic proteins, and we explore these transitions in terms of catalytic chemistry, metabolism, and protein structure. These transitions vary from subtle ones, such as simply repurposing oxidoreductases by replacing an electron acceptor such as NAD by O2 , to drastic changes in reaction mechanism, such as turning carboxylases and hydrolases into oxidases. The latter is more common and can occur with strikingly minor changes, for example, only one mutation in the active site. We further suggest that engineering enzymes to harness the extraordinary reactivity of oxygen may yield higher catabolic power and versatility.

The evolutionary history of the HUP domain.

Among the enzyme lineages that undoubtedly emerged prior to the last universal common ancestor is the so-called HUP, which includes Class I aminoacyl tRNA synthetases (AARSs) as well as enzymes mediating NAD, FAD, and CoA biosynthesis. Here, we provide a detailed analysis of HUP evolution, from emergence to structural and functional diversification. The HUP is a nucleotide binding domain that uniquely catalyzes adenylation via the release of pyrophosphate. In contrast to other ancient nucleotide binding domains with the αßα sandwich architecture, such as P-loop NTPases, the HUP's most conserved feature is not phosphate binding, but rather ribose binding by backbone interactions to the tips of ß1 and/or ß4. Indeed, the HUP exhibits unusual evolutionary plasticity and, while ribose binding is conserved, the location and mode of binding to the base and phosphate moieties of the nucleotide, and to the substrate(s) reacting with it, have diverged with time, foremost along the emergence of the AARSs. The HUP also beautifully demonstrates how a well-packed scaffold combined with evolvable surface elements promotes evolutionary innovation. Finally, we offer a scenario for the emergence of the HUP from a seed ßαß fragment, and suggest that despite an identical architecture, the HUP and the Rossmann represent independent emergences.

The evolution of oxygen-utilizing enzymes suggests early biosphere oxygenation.

Production of molecular oxygen was a turning point in the Earth's history. The geological record indicates the Great Oxidation Event, which marked a permanent transition to an oxidizing atmosphere around 2.4 Ga. However, the degree to which oxygen was available to life before oxygenation of the atmosphere remains unknown. Here, phylogenetic analysis of all known oxygen-utilizing and -producing enzymes (O2-enzymes) indicates that oxygen became widely available to living organisms well before the Great Oxidation Event. About 60% of the O2-enzyme families whose birth can be dated appear to have emerged at the separation of terrestrial and marine bacteria (22 families, compared to two families assigned to the last universal common ancestor). This node, dubbed the last universal oxygen ancestor, coincides with a burst of emergence of both oxygenases and other oxidoreductases, thus suggesting a wider availability of oxygen around 3.1 Ga.

On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment.

This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We detected homologous segments that span the first ßαß motif of both lineages, including the phosphate binding loop and a conserved aspartate at the tip of ß2. The latter ligates the catalytic metal in P-loop NTPases, while in Rossmanns it binds the nucleotide's ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the ß2-Asp to take either one of these two roles. While convergence cannot be completely ruled out, we show that both lineages likely emerged from a common ßαß segment that comprises the core of these enzyme families to this very day.

Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity.

CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast-carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form-II and form-II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s-1 -sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.

Correction to: Classification, substrate specificity and structural features of D-2-hydroxyacid dehydrogenases: 2HADH knowledgebase.

Following publication of the original article [1], we have been notified that some important information was omitted by the authors from the Competing interests section. The declaration should read as below.

The number and type of oxygen-utilizing enzymes indicates aerobic vs. anaerobic phenotype.

Oxygen is a major metabolic driving force that enabled the expansion of metabolic networks including new metabolites and new enzymes. It had a dramatic impact on the primary electron transport chain where it serves as terminal electron acceptor, but oxygen is also used by many enzymes as electron acceptor for a variety of reactions. The organismal oxygen phenotype, aerobic vs. anaerobic, should be manifested in its O2-utilizing enzymes. Traditionally, enzymes involved in primary oxygen metabolism such as cytochrome c, and reactive oxygen species (ROS)-neutralizing enzymes (e.g. catalase), were used as identifiers of oxygen phenotype. However, these enzymes are often found in strict anaerobes. We aimed to identify the O2-utilizing enzymes that may distinguish between aerobes and anaerobes. To this end, we annotated the O2-utilizing enzymes across the prokaryotic tree of life. We recovered over 700 enzymes and mapped their presence/absence in 272 representative genomes. As seen before, enzymes mediating primary oxygen metabolism, and ROS neutralizing enzymes, could be found in both aerobes and anaerobes. However, there exists a subset of enzymes, primarily oxidases that catabolyze various substrates, including amino acids and xenobiotics, that are preferentially enriched in aerobes. Overall it appears that the total number of oxygen-utilizing enzymes, and the presence of enzymes involved in 'peripheral', secondary oxygen metabolism, can reliably distinguish aerobes from anaerobes based solely on genome sequences. These criteria can also indicate the oxygen phenotype in metagenomic samples.

Classification, substrate specificity and structural features of D-2-hydroxyacid dehydrogenases: 2HADH knowledgebase.

BACKGROUND: The family of D-isomer specific 2-hydroxyacid dehydrogenases (2HADHs) contains a wide range of oxidoreductases with various metabolic roles as well as biotechnological applications. Despite a vast amount of biochemical and structural data for various representatives of the family, the long and complex evolution and broad sequence diversity hinder functional annotations for uncharacterized members. RESULTS: We report an in-depth phylogenetic analysis, followed by mapping of available biochemical and structural data on the reconstructed phylogenetic tree. The analysis suggests that some subfamilies comprising enzymes with similar yet broad substrate specificity profiles diverged early in the evolution of 2HADHs. Based on the phylogenetic tree, we present a revised classification of the family that comprises 22 subfamilies, including 13 new subfamilies not studied biochemically. We summarize characteristics of the nine biochemically studied subfamilies by aggregating all available sequence, biochemical, and structural data, providing comprehensive descriptions of the active site, cofactor-binding residues, and potential roles of specific structural regions in substrate recognition. In addition, we concisely present our analysis as an online 2HADH enzymes knowledgebase. CONCLUSIONS: The knowledgebase enables navigation over the 2HADHs classification, search through collected data, and functional predictions of uncharacterized 2HADHs. Future characterization of the new subfamilies may result in discoveries of enzymes with novel metabolic roles and with properties beneficial for biotechnological applications.

A Bird's-Eye View of Enzyme Evolution: Chemical, Physicochemical, and Physiological Considerations.

Enzymes catalyze a vast range of reactions. Their catalytic performances, mechanisms, global folds, and active-site architectures are also highly diverse, suggesting that enzymes are shaped by an entire range of physiological demands and evolutionary constraints, as well as by chemical and physicochemical constraints. We have attempted to identify signatures of these shaping demands and constraints. To this end, we describe a bird's-eye view of the enzyme space from two angles: evolution and chemistry. We examine various chemical reaction parameters that may have shaped the catalytic performances and active-site architectures of enzymes. We test and weigh these considerations against physiological and evolutionary factors. Although the catalytic properties of the "average" enzyme correlate with cellular metabolic demands and enzyme expression levels, at the level of individual enzymes, a multitude of physiological demands and constraints, combined with the coincidental nature of evolutionary processes, result in a complex picture. Indeed, neither reaction type (a chemical constraint) nor evolutionary origin alone can explain enzyme rates. Nevertheless, chemical constraints are apparent in the convergence of active-site architectures in independently evolved enzymes, although significant variations within an architecture are common.

PDBsum: Structural summaries of PDB entries.

PDBsum is a web server providing structural information on the entries in the Protein Data Bank (PDB). The analyses are primarily image-based and include protein secondary structure, protein-ligand and protein-DNA interactions, PROCHECK analyses of structural quality, and many others. The 3D structures can be viewed interactively in RasMol, PyMOL, and a JavaScript viewer called 3Dmol.js. Users can upload their own PDB files and obtain a set of password-protected PDBsum analyses for each. The server is freely accessible to all at: http://www.ebi.ac.uk/pdbsum.

Systematic classification of the His-Me finger superfamily.

The His-Me finger endonucleases, also known as HNH or ßßα-metal endonucleases, form a large and diverse protein superfamily. The His-Me finger domain can be found in proteins that play an essential role in cells, including genome maintenance, intron homing, host defense and target offense. Its overall structural compactness and non-specificity make it a perfectly-tailored pathogenic module that participates on both sides of inter- and intra-organismal competition. An extremely low sequence similarity across the superfamily makes it difficult to identify and classify new His-Me fingers. Using state-of-the-art distant homology detection methods, we provide an updated and systematic classification of His-Me finger proteins. In this work, we identified over 100 000 proteins and clustered them into 38 groups, of which three groups are new and cannot be found in any existing public domain database of protein families. Based on an analysis of sequences, structures, domain architectures, and genomic contexts, we provide a careful functional annotation of the poorly characterized members of this superfamily. Our results may inspire further experimental investigations that should address the predicted activity and clarify the potential substrates, to provide more detailed insights into the fundamental biological roles of these proteins.

SupeRNAlign: a new tool for flexible superposition of homologous RNA structures and inference of accurate structure-based sequence alignments.

RNA has been found to play an ever-increasing role in a variety of biological processes. The function of most non-coding RNA molecules depends on their structure. Comparing and classifying macromolecular 3D structures is of crucial importance for structure-based function inference and it is used in the characterization of functional motifs and in structure prediction by comparative modeling. However, compared to the numerous methods for protein structure superposition, there are few tools dedicated to the superimposing of RNA 3D structures. Here, we present SupeRNAlign (v1.3.1), a new method for flexible superposition of RNA 3D structures, and SupeRNAlign-Coffee-a workflow that combines SupeRNAlign with T-Coffee for inferring structure-based sequence alignments. The methods have been benchmarked with eight other methods for RNA structural superposition and alignment. The benchmark included 151 structures from 32 RNA families (with a total of 1734 pairwise superpositions). The accuracy of superpositions was assessed by comparing structure-based sequence alignments to the reference alignments from the Rfam database. SupeRNAlign and SupeRNAlign-Coffee achieved significantly higher scores than most of the benchmarked methods: SupeRNAlign generated the most accurate sequence alignments among the structure superposition methods, and SupeRNAlign-Coffee performed best among the sequence alignment methods.

cgmisc: enhanced genome-wide association analyses and visualization.

UNLABELLED: High-throughput genotyping and sequencing technologies facilitate studies of complex genetic traits and provide new research opportunities. The increasing popularity of genome-wide association studies (GWAS) leads to the discovery of new associated loci and a better understanding of the genetic architecture underlying not only diseases, but also other monogenic and complex phenotypes. Several softwares are available for performing GWAS analyses, R environment being one of them. RESULTS: We present cgmisc, an R package that enables enhanced data analysis and visualization of results from GWAS. The package contains several utilities and modules that complement and enhance the functionality of the existing software. It also provides several tools for advanced visualization of genomic data and utilizes the power of the R language to aid in preparation of publication-quality figures. Some of the package functions are specific for the domestic dog (Canis familiaris) data. AVAILABILITY AND IMPLEMENTATION: The package is operating system-independent and is available from: https://github.com/cgmisc-team/cgmisc CONTACT: marcin.kierczak@imbim.uu.se. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.