Enzyme function and evolution through the lens of bioinformatics.

Enzymes have been shaped by evolution over billions of years to catalyse the chemical reactions that support life on earth. Dispersed in the literature, or organised in online databases, knowledge about enzymes can be structured in distinct dimensions, either related to their quality as biological macromolecules, such as their sequence and structure, or related to their chemical functions, such as the catalytic site, kinetics, mechanism, and overall reaction. The evolution of enzymes can only be understood when each of these dimensions is considered. In addition, many of the properties of enzymes only make sense in the light of evolution. We start this review by outlining the main paradigms of enzyme evolution, including gene duplication and divergence, convergent evolution, and evolution by recombination of domains. In the second part, we overview the current collective knowledge about enzymes, as organised by different types of data and collected in several databases. We also highlight some increasingly powerful computational tools that can be used to close gaps in understanding, in particular for types of data that require laborious experimental protocols. We believe that recent advances in protein structure prediction will be a powerful catalyst for the prediction of binding, mechanism, and ultimately, chemical reactions. A comprehensive mapping of enzyme function and evolution may be attainable in the near future.

EzMechanism: an automated tool to propose catalytic mechanisms of enzyme reactions.

Over the years, hundreds of enzyme reaction mechanisms have been studied using experimental and simulation methods. This rich literature on biological catalysis is now ripe for use as the foundation of new knowledge-based approaches to investigate enzyme mechanisms. Here, we present a tool able to automatically infer mechanistic paths for a given three-dimensional active site and enzyme reaction, based on a set of catalytic rules compiled from the Mechanism and Catalytic Site Atlas, a database of enzyme mechanisms. EzMechanism (pronounced as 'Easy' Mechanism) is available to everyone through a web user interface. When studying a mechanism, EzMechanism facilitates and improves the generation of hypotheses, by making sure that relevant information is considered, as derived from the literature on both related and unrelated enzymes. We validated EzMechanism on a set of 62 enzymes and have identified paths for further improvement, including the need for additional and more generic catalytic rules.

The 3D Modules of Enzyme Catalysis: Deconstructing Active Sites into Distinct Functional Entities.

Enzyme catalysis is governed by a limited toolkit of residues and organic or inorganic co-factors. Therefore, it is expected that recurring residue arrangements will be found across the enzyme space, which perform a defined catalytic function, are structurally similar and occur in unrelated enzymes. Leveraging the integrated information in the Mechanism and Catalytic Site Atlas (M-CSA) (enzyme structure, sequence, catalytic residue annotations, catalysed reaction, detailed mechanism description), 3D templates were derived to represent compact groups of catalytic residues. A fuzzy template-template search, allowed us to identify those recurring motifs, which are conserved or convergent, that we define as the "modules of enzyme catalysis". We show that a large fraction of these modules facilitate binding of metal ions, co-factors and substrates, and are frequently the result of convergent evolution. A smaller number of convergent modules perform a well-defined catalytic role, such as the variants of the catalytic triad (i.e. Ser-His-Asp/Cys-His-Asp) and the saccharide-cleaving Asp/Glu triad. It is also shown that enzymes whose functions have diverged during evolution preserve regions of their active site unaltered, as shown by modules performing similar or identical steps of the catalytic mechanism. We have compiled a comprehensive library of catalytic modules, that characterise a broad spectrum of enzymes. These modules can be used as templates in enzyme design and for better understanding catalysis in 3D.

Modification of chicha gum: Antibacterial activity, ex vivo mucoadhesion, antioxidant activity and cellular viability.

The aim of the present work was to modify the exuded gum of Sterculia striata tree by an amination reaction. The viscosity and zero potential of the chicha gum varied as a function of pH. The modification was confirmed by X-ray diffraction (XRD), infrared spectroscopy (FTIR), size exclusion chromatography (SEC), zeta potential, thermogravimetric analysis (TG), and differential scanning calorimetry (DSC). Furthermore, the chemical modification changed the molar mass and surface charge of the chicha gum. In addition, the gums were used in tests for ex vivo mucoadhesion strength, antibacterial activity against the standard strain of Staphylococcus aureus (ATCC 25923), inhibitory activity of α-glucosidase, antioxidant capacity, and viability of Caco-2 cells. Through these tests, it was found that amination caused an increase in the mucoadhesive and inhibitory activity of chicha gum against the bacterium Staphylococcus aureus. In addition, the gums (pure and modified) showed antioxidant capacity and an inhibitory effect against the α-glucosidase enzyme and did not show cytotoxic potential.

Lipoplexes and polyplexes as nucleic acids delivery nanosystems: The current state and future considerations.

INTRODUCTION: Designing safe and effective nucleic acid delivery nanosystems presents a challenge that requires a good understanding of various biological barriers, whose impact is frequently neglected during in vitro assessments. Hence, the development of nanosizing non-viral vectors would benefit from a more thorough physicochemical characterization to establish structure-activity relationships and increase the preclinical data relevance. AREAS COVERED: This review focused on major barriers of lipoplexes and polyplexes by systemic delivery such as blood and immune cells and is aimed to serve as a prescreening tool for the fast and safe development of both non-viral vectors in vivo. An outline of the preclinical assays to be performed under physiological representative conditions, to better account for or even predict the highly dynamic interactions in humans is also given. EXPERT OPINION: The rational design of non-viral vectors has shown promising intracellular uptake results in vitro. Translating in vitro success into clinics has gone with progress, but it is still a difficult task to achieve, and more closely mimicking biological environment in vitro assays of lipoplexes and polyplexes may provide more correlated results to in vivo experiments. Clinical practice would benefit from safer non-viral vectors, particularly when avoiding patients' immune responses and toxicity, which is of major concern.

GRaSP-web: a machine learning strategy to predict binding sites based on residue neighborhood graphs.

Proteins are essential macromolecules for the maintenance of living systems. Many of them perform their function by interacting with other molecules in regions called binding sites. The identification and characterization of these regions are of fundamental importance to determine protein function, being a fundamental step in processes such as drug design and discovery. However, identifying such binding regions is not trivial due to the drawbacks of experimental methods, which are costly and time-consuming. Here we propose GRaSP-web, a web server that uses GRaSP (Graph-based Residue neighborhood Strategy to Predict binding sites), a residue-centric method based on graphs that uses machine learning to predict putative ligand binding site residues. The method outperformed 6 state-of-the-art residue-centric methods (MCC of 0.61). Also, GRaSP-web is scalable as it takes 10-20 seconds to predict binding sites for a protein complex (the state-of-the-art residue-centric method takes 2-5h on the average). It proved to be consistent in predicting binding sites for bound/unbound structures (MCC 0.61 for both) and for a large dataset of multi-chain proteins (4500 entries, MCC 0.61). GRaSPWeb is freely available at https://grasp.ufv.br.

Conformational Variation in Enzyme Catalysis: A Structural Study on Catalytic Residues.

Conformational variation in catalytic residues can be captured as alternative snapshots in enzyme crystal structures. Addressing the question of whether active site flexibility is an intrinsic and essential property of enzymes for catalysis, we present a comprehensive study on the 3D variation of active sites of 925 enzyme families, using explicit catalytic residue annotations from the Mechanism and Catalytic Site Atlas and structural data from the Protein Data Bank. Through weighted pairwise superposition of the functional atoms of active sites, we captured structural variability at single-residue level and examined the geometrical changes as ligands bind or as mutations occur. We demonstrate that catalytic centres of enzymes can be inherently rigid or flexible to various degrees according to the function they perform, and structural variability most often involves a subset of the catalytic residues, usually those not directly involved in the formation or cleavage of bonds. Moreover, data suggest that 2/3 of active sites are flexible, and in half of those, flexibility is only observed in the side chain. The goal of this work is to characterise our current knowledge of the extent of flexibility at the heart of catalysis and ultimately place our findings in the context of the evolution of catalysis as enzymes evolve new functions and bind different substrates.

The Enzyme Portal: an integrative tool for enzyme information and analysis.

Enzymes play essential roles in all life processes and are used extensively in the biomedical and biotechnological fields. However, enzyme-related information is spread across multiple resources making its retrieval time-consuming. In response to this challenge, the Enzyme Portal has been established to facilitate enzyme research, by providing a freely available hub where researchers can easily find and explore enzyme-related information. It integrates relevant enzyme data for a wide range of species from various resources such as UniProtKB, PDBe and ChEMBL. Here, we describe what type of enzyme-related data the Enzyme Portal provides, how the information is organized and, by show-casing two potential use cases, how to access and retrieve it.

Using mechanism similarity to understand enzyme evolution.

Enzyme reactions take place in the active site through a series of catalytic steps, which are collectively termed the enzyme mechanism. The catalytic step is thereby the individual unit to consider for the purposes of building new enzyme mechanisms - i.e. through the mix and match of individual catalytic steps, new enzyme mechanisms and reactions can be conceived. In the case of natural evolution, it has been shown that new enzyme functions have emerged through the tweaking of existing mechanisms by the addition, removal, or modification of some catalytic steps, while maintaining other steps of the mechanism intact. Recently, we have extracted and codified the information on the catalytic steps of hundreds of enzymes in a machine-readable way, with the aim of automating this kind of evolutionary analysis. In this paper, we illustrate how these data, which we called the "rules of enzyme catalysis", can be used to identify similar catalytic steps across enzymes that differ in their overall function and/or structural folds. A discussion on a set of three enzymes that share part of their mechanism is used as an exemplar to illustrate how this approach can reveal divergent and convergent evolution of enzymes at the mechanistic level. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-022-01022-9.

GRaSP: a graph-based residue neighborhood strategy to predict binding sites.

MOTIVATION: The discovery of protein-ligand-binding sites is a major step for elucidating protein function and for investigating new functional roles. Detecting protein-ligand-binding sites experimentally is time-consuming and expensive. Thus, a variety of in silico methods to detect and predict binding sites was proposed as they can be scalable, fast and present low cost. RESULTS: We proposed Graph-based Residue neighborhood Strategy to Predict binding sites (GRaSP), a novel residue centric and scalable method to predict ligand-binding site residues. It is based on a supervised learning strategy that models the residue environment as a graph at the atomic level. Results show that GRaSP made compatible or superior predictions when compared with methods described in the literature. GRaSP outperformed six other residue-centric methods, including the one considered as state-of-the-art. Also, our method achieved better results than the method from CAMEO independent assessment. GRaSP ranked second when compared with five state-of-the-art pocket-centric methods, which we consider a significant result, as it was not devised to predict pockets. Finally, our method proved scalable as it took 10-20 s on average to predict the binding site for a protein complex whereas the state-of-the-art residue-centric method takes 2-5 h on average. AVAILABILITY AND IMPLEMENTATION: The source code and datasets are available at https://github.com/charles-abreu/GRaSP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Sterculia striata gum as a potential oral delivery system for protein drugs.

Natural polysaccharides have been investigated as vehicles for oral insulin administration. Because of their non-toxic, renewable, low cost and readily available properties, gums find multiple applications in the pharmaceutical industry. This work aimed to develop a Sterculia striata gum-based formulation associated with additional biopolymers (dextran sulfate, chitosan, and albumin), a crosslinking agent (calcium chloride) and stabilizing agents (polyethylene glycol and poloxamer 188), to increase the oral bioavailability of proteins. Insulin was used as a model drug and the methods used to prepare the formulation were based on ionotropic pregelation followed by electrolytic complexation of oppositely charged biopolymers under controlled pH conditions. The developed formulation was characterized to validate its efficacy, by the determination of its average particle size (622 nm), the insulin encapsulation efficiency (70%), stability in storage for 30 days, and the in vitro mucoadhesion strength (92.46 mN). Additionally, the developed formulation preserved about 64% of initial insulin dose in a simulated gastric medium. This study proposed, for the first time, a Sterculia striata gum-based insulin delivery system with potential for the oral administration of protein drugs, being considered a valid alternative for efficient delivery of those drugs.

Identifying pseudoenzymes using functional annotation: pitfalls of common practice.

Pseudoenzymes are proteins that are evolutionary related to enzymes but lack relevant catalytic activity. They are usually evolved from enzymatic ancestors that have lost their catalytic activities. The loss of catalytic function is one extreme amongst the other evolutionary changes that can occur to enzymes, like the changing of substrate specificity or the reaction catalysed. However, the loss of catalytic function events remain poorly characterised, except for some notable examples, like the pseudokinases. In this review, we aim to analyse current knowledge related to pseudoenzymes across a large number of enzymes families. This aims to be a review of the data available in biological databases, rather than a more traditional literature review. In particular, we use UniProtKB as the source for functional annotation and M-CSA (Mechanism and Catalytic Site Atlas) for information on the catalytic residues of enzymes. We show that explicit annotation of lack of activity is not exhaustive in UniProtKB and that a protocol using lack of catalytic annotation as an indication for lack of function can be an adequate alternative, after some corrections. After identifying pseudoenzymes related to enzymes in M-CSA, we were able to comment on their prevalence across enzyme families, and on the correlation between lack of catalytic function and the mutation of catalytic residues. These analyses challenge two common ideas in the emerging literature: that pseudoenzymes are ubiquitous across enzyme families and that mutations in the catalytic residues of enzyme homologues are always a good indication of lack of activity.

A global analysis of function and conservation of catalytic residues in enzymes.

The catalytic residues of an enzyme comprise the amino acids located in the active center responsible for accelerating the enzyme-catalyzed reaction. These residues lower the activation energy of reactions by performing several catalytic functions. Decades of enzymology research has established general themes regarding the roles of specific residues in these catalytic reactions, but it has been more difficult to explore these roles in a more systematic way. Here, we review the data on the catalytic residues of 648 enzymes, as annotated in the Mechanism and Catalytic Site Atlas (M-CSA), and compare our results with those in previous studies. We structured this analysis around three key properties of the catalytic residues: amino acid type, catalytic function, and sequence conservation in homologous proteins. As expected, we observed that catalysis is mostly accomplished by a small set of residues performing a limited number of catalytic functions. Catalytic residues are typically highly conserved, but to a smaller degree in homologues that perform different reactions or are nonenzymes (pseudoenzymes). Cross-analysis yielded further insights revealing which residues perform particular functions and how often. We obtained more detailed specificity rules for certain functions by identifying the chemical group upon which the residue acts. Finally, we show the mutation tolerance of the catalytic residues based on their roles. The characterization of the catalytic residues, their functions, and conservation, as presented here, is key to understanding the impact of mutations in evolution, disease, and enzyme design. The tools developed for this analysis are available at the M-CSA website and allow for user specific analysis of the same data.

Exploring Chemical Biosynthetic Design Space with Transform-MinER.

Transform-MinER (Transforming Molecules in Enzyme Reactions) is a web application facilitating the exploration of chemical biosynthetic space, guiding the user toward promising start points for enzyme design projects or directed evolution experiments. Two types of search are possible: Molecule Search allows a user to submit a source substrate enabling Transform-MinER to search for enzyme reactions acting on similar substrates, whereas Path Search additionally allows a user to submit a target molecule enabling Transform-MinER to search for a path of enzyme reactions acting on similar substrates to link source and target. Transform-MinER searches for potential reaction centers in the source substrate and uses chemoinformatic fingerprints to identify those that are situated in molecular environments similar to native counterparts, prioritizing steps that move closer to the target using reactions most similar to native in its exploration of search space. The ligand-based methodology behind Transform-MinER is presented, and its performance is validated yielding 90% success rates: first, on a data set of native pathways from the KEGG database, and second, on a data set of de novo enzyme reactions.

Sonication-assisted Layer-by-Layer self-assembly nanoparticles for resveratrol delivery.

This paper advances the development of a novel drug nanodelivery solution to the oral administration of resveratrol (RSV), a low soluble drug whose recognized therapeutic applications are circumscribed when administered in the free compound form. Layer-by-Layer (LbL) self-assembly is an emergent nanotechnology proposed to address this concern with means to afford control over key formulation parameters, which are able to ultimately promote an improved pharmacokinetics. LbL self-assembly consists in the sequential adsorption of oppositely charged polyelectrolytes upon a low soluble drug nanoparticle (NP) template, giving rise to onion-like multilayered nanoarchitectures. In this work, RSV nanoprecipitation followed by LbL self-assembly of polyelectrolytes, led by a washless approach, was carried out by using the cationic poly(allylamine hydrochloride) (PAH) and the anionic dextran sulfate (DS) as polyelectrolytes towards the nanoencapsulation of RSV. Each saturated polyelectrolyte layer deposition involved the rigorous polyelectrolyte concentration assessment which was accomplished by tracing titration curves. This way, aqueous RSV nanocores and RSV LbL nanoformulations with a distinct number of PAH/DS bilayers were developed, including 2.5 (RSV-(PAH/DS)2.5 NPs), 5.5 (RSV-(PAH/DS)5.5 NPs) and 7.5 (RSV-(PAH/DS)7.5 NPs) bilayered nanoformulations. Homogenous particle size distributions at the desired nanoscale interval (ca. 116-220 nm; polydispersity index below 0.15), good colloidal (zeta potential magnitudes ca. ± 20-30 mV) and chemical stabilizations, high encapsulation efficiency (above 90%) together with an excellent cytocompatibility with Caco-2 cells (cell viability above 90%) were observed for all the nanoformulations. Eventfully, LbL NPs promoted a controlled release of RSV pursuant to the number of polyelectrolyte bilayers under simulated gastrointestinal conditions, particularly in the intestine medium, emphasizing their biopharmaceutical advantage. Our findings manifestly pinpoint that LbL PAH/DS NPs constitute a promising nanodelivery system for the oral delivery of RSV, providing a rational strategy to enlarge the implementation range of this interesting polyphenol, which is possibly the most actively investigated phytochemical worldwide.

Emerging concepts in pseudoenzyme classification, evolution, and signaling.

The 21st century is witnessing an explosive surge in our understanding of pseudoenzyme-driven regulatory mechanisms in biology. Pseudoenzymes are proteins that have sequence homology with enzyme families but that are proven or predicted to lack enzyme activity due to mutations in otherwise conserved catalytic amino acids. The best-studied pseudoenzymes are pseudokinases, although examples from other families are emerging at a rapid rate as experimental approaches catch up with an avalanche of freely available informatics data. Kingdom-wide analysis in prokaryotes, archaea and eukaryotes reveals that between 5 and 10% of proteins that make up enzyme families are pseudoenzymes, with notable expansions and contractions seemingly associated with specific signaling niches. Pseudoenzymes can allosterically activate canonical enzymes, act as scaffolds to control assembly of signaling complexes and their localization, serve as molecular switches, or regulate signaling networks through substrate or enzyme sequestration. Molecular analysis of pseudoenzymes is rapidly advancing knowledge of how they perform noncatalytic functions and is enabling the discovery of unexpected, and previously unappreciated, functions of their intensively studied enzyme counterparts. Notably, upon further examination, some pseudoenzymes have previously unknown enzymatic activities that could not have been predicted a priori. Pseudoenzymes can be targeted and manipulated by small molecules and therefore represent new therapeutic targets (or anti-targets, where intervention should be avoided) in various diseases. In this review, which brings together broad bioinformatics and cell signaling approaches in the field, we highlight a selection of findings relevant to a contemporary understanding of pseudoenzyme-based biology.

Targeting Cancer Via Resveratrol-Loaded Nanoparticles Administration: Focusing on In Vivo Evidence.

Resveratrol (RSV) is a polyphenol endowed with potential therapeutic effects in chronic diseases, particularly in cancer, the second leading cause of death worldwide in the twenty-first century. The advent of nanotechnology application in the field of drug delivery allows to overcome the constrains associated with the conventional anticancer treatments, in particular chemotherapy, reducing its adverse side effects, off target risks and surpassing cancer multidrug chemoresistance. Moreover, the use of nanotechnology-based carriers in the delivery of plant-derived anticancer agents, such as RSV, has already demonstrated to surpass the poor water solubility, instability and reduced bioavailability associated with phytochemicals, improving their therapeutic activity, thus prompting pharmaceutical developments. This review highlights the in vivo anticancer potential of RSV achieved by nanotherapeutic approaches. First, RSV physicochemical, stability and pharmacokinetic features are described. Thereupon, the chemotherapeutic and chemopreventive properties of RSV are underlined, emphasizing the RSV numerous cancer molecular targets. Lastly, a comprehensive analysis of the RSV-loaded nanoparticles (RSV-NPs) developed and administered in different in vivo cancer models to date is presented. Nanoparticles (NPs) have shown to improve RSV solubility, stability, pharmacokinetics and biodistribution in cancer tissues, enhancing markedly its in vivo anticancer activity. RSV-NPs are, thus, considered a potential nanomedicine-based strategy to fight cancer; however, further studies are still necessary to allow RSV-NP clinical translation.

Nanotechnology-based formulations for resveratrol delivery: Effects on resveratrol in vivo bioavailability and bioactivity.

Resveratrol (RES), also known as 3,5,4'-trihydroxystilbene, is a polyphenolic phytoalexin that has been widely researched in the past decade due to its recognized numerous biological activities. Despite the potential benefits of RES, its effective use is limited due to its poor solubility, photosensitivity and rapid metabolism, which strongly undermine RES bioavailability and bioactivity. Thereby, recently, nanotechnology appeared as a potential strategy to circumvent RES physicochemical and pharmacokinetics constrains. However, only few studies have addressed the crucial in vivo suitability of the developed delivery systems to improve RES efficacy. Facing this scenario, in the present review, it is intended to present and discuss the in vivo resveratrol bioavailability and bioactivity, following its encapsulation or conjugation in nanotechnology-based carriers, contemplating their pharmacokinetics effectiveness.

First-time oral administration of resveratrol-loaded layer-by-layer nanoparticles to rats - a pharmacokinetics study.

trans-Resveratrol (RSV) is a plant-derived polyphenol endowed with a broad spectrum of promising therapeutic activities. The applicability of RSV in vivo has, however, had limited success so far, largely due to its inefficient systemic delivery resulting from its low water solubility. Layer-by-Layer (LbL) nanotechnology constitutes an innovative formulation strategy to address this concern, and is based on the design of tunable onion-like multilayered nanoarchitectures on the surface of low solubility drug nanocores, such as RSV. The purpose of this study was the investigation of the bioavailability of an LbL nanoformulation composed of 5.5 bilayers of polyallylamine hydrochloride (PAH) and dextran sulfate (DS) (LbL NPs) by pharmacokinetic studies following oral dosing to Wistar rats (20 mg kg-1). The systemic exposure of LbL NPs was compared to the respective nanoformulation without LbL coatings (RSV nanocores) and the free RSV suspension. The results demonstrated that both LbL NPs and RSV nanocores significantly enhanced, respectively, 1.76-fold and 2.74-fold the systemic exposure of RSV compared to the free RSV suspension, emphasizing their biopharmaceutical advantage. Surprisingly, besides the modified drug release potential of the LbL NPs, these exhibited a slightly lower systemic exposure (0.36-fold) in comparison with non-LbL modified RSV nanocores. These results were justified only by the electrostatic interactions composition of the LbL shell composition, requiring further research towards the application of stronger interactions. For this study, due to the key role of the bioanalytical method in the in vivo data acquisition, a rapid, selective, and sensitive HPLC-DAD method has been successfully optimized and fully validated to confidently quantify RSV levels in the rat plasma matrix, together with the optimization of the sample preparation procedure. Moreover, the chemical stability of RSV was evaluated for 24 h in simulated gastric and intestinal fluids with enzymes. Overall, our findings suggest that LbL NPs should be given great attention, representing a potential drug delivery system for RSV in view of the application of RSV not solely as a supplement but also as a therapeutic drug.

Subcutaneous delivery of biotherapeutics: challenges at the injection site.

INTRODUCTION: Biotherapeutics are primarily delivered subcutaneously due to better compliance and prolonged rate of absorption compared to other parenteral administration routes. Recent research has allowed for the development of biotherapeutic formulations for subcutaneous delivery that require a lower frequency of administration by increasing drug half-life. Formulations determine shelf-life stability as well as features and transient behaviors that influence stability once implanted in the subcutaneous space. AREAS COVERED: This review provides an overview of the factors affecting subcutaneous absorption with a focus on transient effects at the injection site following administration of biotherapeutics and the subsequent impact on absorption and stability. EXPERT OPINION: Advances have been made in understanding subcutaneous tissue and the complex interplay of factors that regulate its homeostasis. The issue of poor stability after injection has been neglected, and many biotherapeutics are hampered by low bioavailability. With the advent of new in vitro techniques that account for properties of the injection site, stability studies evaluating subcutaneous tissues and impacts on pharmacokinetics of biotherapeutics may be useful in the development of new formulations.