The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called "orphan enzymes". The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to "local orphan enzymes" that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new activities.

No abstract available

Computational prediction of protein function constitutes one of the more complex problems in Bioinformatics, because of the diversity of functions and mechanisms in that proteins exert in nature. This issue is reinforced especially for proteins that share very low primary or tertiary structure similarity to existing annotated proteomes. In this sense, new alignment-free (AF) tools are needed to overcome the inherent limitations of classic alignment-based approaches to this issue. We have recently introduced AF protein-numerical-encoding programs (TI2BioP and ProtDCal), whose sequence-based features have been successfully applied to detect remote protein homologs, post-translational modifications and antibacterial peptides. Here we aim to demonstrate the applicability of 4 AF protein descriptor families, implemented in our programs, for the identification enzyme-like proteins. At the same time, the use of our novel family of 3D–structure-based descriptors is introduced for the first time. The Dobson & Doig (D&D) benchmark dataset is used for the evaluation of our AF protein descriptors, because of its proven structural diversity that permits one to emulate an experiment within the twilight zone of alignment-based methods (pair-wise identity <30%). The performance of our sequence-based predictor was further assessed using a subset of formerly uncharacterized proteins which currently represent a benchmark annotation dataset.

Enzymes called Rab GTPases that carry so-called "activating" mutations may never become activated at all.

This paper describes a database for cell signaling enzymes. Our web database offers methods to study, interpret and compare cell-signaling enzymes. Searching and retrieving data from this database has been made easy and user friendly and it is well integrated with other related databases. We believe the end user will be benefited from this database.

We describe a general method for the entrapment of enzymes within bulk metallic gold. This is a new approach for the immobilization of enzymes on metals, which is commonly carried out by 2D adsorption or covalent biding, that is, the enzyme is in contact with the metal at a specific contact zone of the enzyme, while most of the rest of it remains exposed to the environment. The 3D metallic encaging of the enzymes is quite different: the enzyme is in contact with the metallic cage walls all around it and is well protected inside. The porous nature of the metallic matrix enables substrate molecules to diffuse inside, reach the active site, and let product molecules diffuse out. The generality of the approach was proven by the successful entrapment of five enzymes representing different classes and different bio- and medical applications: l-asparaginase (Asp), collagenase, horseradish peroxidase (HRP), laccase and glucose oxidase (GOx). GOx-gold conjugates have been of particular interest in the literature. The main challenge we had to solve was how to keep the enzyme active in the process of gold-synthesis from its cation - this required careful tailoring of reaction conditions, which are detailed in the paper. The gold entrapped enzymes gain thermal stability and protectability against harsh conditions. For instance, we could keep Asp alive at the extreme pH of 13, which normally kills the enzyme instantly. The entrapped enzymes obey the Michaelis-Menten kinetics, and activation energies were determined. Good recyclability for eight cycles was found. Multi-enzymatic reactions by combinations of the off-the-shelf bioactive enzyme@gold powders are possible, as demonstrated for the classical detection of GOx activity with HRP. Detailed material characterization and proposed mechanisms for the 3D protectability of the enzymes are provided. The new enzyme immobilization method is of wide potential uses in medicine, biotechnology, bio-fuel cells and enzymatic (electro)sensing applications.

Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration-a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2'-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding.

The ability to alter the genomes of living cells is key to understanding how genes influence the functions of organisms and will be critical to modify living systems for useful purposes. However, this promise has long been limited by the technical challenges involved in genetic engineering. Recent advances in gene editing have bypassed some of these challenges but they are still far from ideal. Here we use FuncLib to computationally design Cas9 enzymes with substantially higher donor-independent editing activities. We use genetic circuits linked to cell survival in yeast to quantify Cas9 activity and discover synergistic interactions between engineered regions. These hyperactive Cas9 variants function efficiently in mammalian cells and introduce larger and more diverse pools of insertions and deletions into targeted genomic regions, providing tools to enhance and expand the possible applications of CRISPR-based gene editing.

The diverse roles of deubiquitinating enzymes, or DUBs, in determining the fate of specific proteins continue to unfold. Concurrent with the revelation of DUBs as potential therapeutic targets are publications of small molecule inhibitors of these enzymes. In this review, we summarize these molecules and their associated data and suggest additional experiments to further validate and characterize these compounds. We believe the field of drug discovery against DUBs is still in its infancy, but advances in assay development, biophysical techniques, and screening libraries hold promise for identifying suitable agents that could advance into the clinic.

Enzymatic degradation of plant biomass has attracted intensive research interest for the production of economically viable biofuels. Here we present an overview of the recent findings on biocatalysts implicated in the oxidative cleavage of cellulose, including polysaccharide monooxygenases (PMOs or LPMOs which stands for lytic PMOs), cellobiose dehydrogenases (CDHs) and members of carbohydrate-binding module family 33 (CBM33). PMOs, a novel class of enzymes previously termed GH61s, boost the efficiency of common cellulases resulting in increased hydrolysis yields while lowering the protein loading needed. They act on the crystalline part of cellulose by generating oxidized and non-oxidized chain ends. An external electron donor is required for boosting the activity of PMOs. We discuss recent findings concerning their mechanism of action and identify issues and questions to be addressed in the future.

Using a microscopic theory to analyze experiments, we demonstrate that enzymes are active matter. Superresolution fluorescence measurements-performed across four orders of magnitude of substrate concentration, with emphasis on the biologically relevant regime around or below the Michaelis-Menten constant-show that catalysis boosts the motion of enzymes to be superdiffusive for a few microseconds, enhancing their effective diffusivity over longer timescales. Occurring at the catalytic turnover rate, these fast ballistic leaps maintain direction over a duration limited by rotational diffusion, driving enzymes to execute wormlike trajectories by piconewton forces performing work of a few kBT against viscosity. The boosts are more frequent at high substrate concentrations, biasing the trajectories toward substrate-poor regions, thus exhibiting antichemotaxis, demonstrated here experimentally over a wide range of aqueous concentrations. Alternative noncatalytic, passive mechanisms that predict chemotaxis, cross-diffusion, and phoresis, are critically analyzed. We examine the physical interpretation of our findings, speculate on the underlying mechanism, and discuss the avenues they open with biological and technological implications. These findings violate the classical paradigm that chemical reaction and motility are distinct processes, and suggest reaction-motion coupling as a general principle of catalysis.

Lignocellulosic biomass is the most abundant source of carbon-based material on a global basis, serving as a raw material for cellulosic fibers, hemicellulosic polymers, platform sugars, and lignin resins or monomers. In nature, the various components of lignocellulose (primarily cellulose, hemicellulose, and lignin) are decomposed by saprophytic fungi and bacteria utilizing specialized enzymes. Enzymes are specific catalysts and can, in many cases, be produced on-site at lignocellulose biorefineries. In addition to reducing the use of often less environmentally friendly chemical processes, the application of such enzymes in lignocellulose processing to obtain a range of specialty products can maximize the use of the feedstock and valorize many of the traditionally underutilized components of lignocellulose, while increasing the economic viability of the biorefinery. While cellulose has a rich history of use in the pulp and paper industries, the hemicellulosic fraction of lignocellulose remains relatively underutilized in modern biorefineries, among other reasons due to the heterogeneous chemical structure of hemicellulose polysaccharides, the composition of which varies significantly according to the feedstock and the choice of pretreatment method and extraction solvent. This paper reviews the potential of hemicellulose in lignocellulose processing with focus on what can be achieved using enzymatic means. In particular, we discuss the various enzyme activities required for complete depolymerization of the primary hemicellulose types found in plant cell walls and for the upgrading of hemicellulosic polymers, oligosaccharides, and pentose sugars derived from hemicellulose depolymerization into a broad spectrum of value-added products.

Despite reports implicating disrupted purine metabolism in causing a wide spectrum of neurological defects, the mechanistic details of purine biosynthesis in neurons are largely unknown. As an initial step in filling that gap, we examined the expression and subcellular distribution of three purine biosynthesis enzymes (PFAS, PAICS and ATIC) in rat hippocampal neurons. Using immunoblotting and high-resolution light and electron microscopic analysis, we find that all three enzymes are broadly distributed in hippocampal neurons with pools of these enzymes associated with mitochondria. These findings suggest a potential link between purine metabolism and mitochondrial function in neurons and provide an impetus for further studies.

The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degradation as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the molecular changes that led to the appearance of the same surface oxidation site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin.

Scedosporium species are usually soil saprophytes but some members of the genus such as S. apiospermum and S. aurantiacum have been regularly reported as causing human respiratory infections, particularly in patients with cystic fibrosis (CF). Because of their low sensitivity to almost all available antifungal drugs, a better understanding of the pathogenic mechanisms of these fungi is mandatory. Likewise, identification of the origin of the contamination of patients with CF may be helpful to propose prophylactic measures. In this aim, environmental studies were conducted demonstrating that Scedosporium species are abundant in human-made environments and associated with nutrient-rich substrates. Although their natural habitat remains unknown, there is accumulated evidence to consider them as wood-decaying fungi. This study aimed to demonstrate the ability of these fungi to utilize lignocellulose compounds, especially lignin, as a carbon source. First, the lignolytic properties of Scedosporium species were confirmed by cultural methods, and biochemical assays suggested the involvement of peroxidases and oxidases as lignin-modifying enzymes. Scedosporium genomes were then screened using tBLASTn searches. Fifteen candidate genes were identified, including four peroxidase and seven oxidase genes, and some of them were shown, by real-time PCR experiments, to be overexpressed in lignin-containing medium, thus confirming their involvement in lignin degradation.

The adhesion forces have been measured between an atomic force microscope tip derivatized with an active enzyme, shikimate kinase, and an ATP mimic immobilized on a gold surface. Experiments with competitive binding of other ligands in solution show that the observed adhesion forces arise predominantly from specific interactions between the immobilized enzyme and surface-bound adenine derivative. These experiments represent a step in the development of a screening methodology based upon chemical force microscopy.

Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass.

Ether lipids are an emerging class of lipids which have so far not been investigated and understood in every detail. They have important roles as membrane components of e.g. lens, brain and testis, and as mediators such as platelet-activating factor. The metabolic enzymes for biosynthesis and degradation have been investigated to some extent. As most involved enzymes are integral membrane proteins they are tricky to handle in biochemical protocols. The sequence of some ether lipid metabolising enzymes has only recently been reported and other sequences still remain obscure. Defined enzymes without assigned sequence are known as orphan enzymes. One of these enzymes with uncharacterised sequence is plasmanylethanolamine desaturase, a key enzyme for the biosynthesis of one of the most abundant phospholipids in our body, the plasmalogens. This review aims to briefly summarise known functions of ether lipids, give an overview on their metabolism including the most prominent members, platelet-activating factor and the plasmalogens. A special focus is set on the description of orphan enzymes in ether lipid metabolism and on the successful strategies how four previous orphans have recently been assigned a sequence. Only one of these four was characterised by classical protein purification and sequencing, whereas the other three required alternative strategies such as bioinformatic candidate gene selection and recombinant expression or development of an inhibitor and multidimensional metabolic profiling.

The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism's fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.

Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.