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Thirty years have elapsed since the emergence of the classification of carbohydrate-active enzymes in sequence-based families that became the CAZy database over 20 years ago, freely available for browsing and download at www.cazy.org. In the era of large scale sequencing and high-throughput Biology, it is important to examine the position of this specialist database that is deeply rooted in human curation. The three primary tasks of the CAZy curators are (i) to maintain and update the family classification of this class of enzymes, (ii) to classify sequences newly released by GenBank and the Protein Data Bank and (iii) to capture and present functional information for each family. The CAZy website is updated once a month. Here we briefly summarize the increase in novel families and the annotations conducted during the last 8 years. We present several important changes that facilitate taxonomic navigation, and allow to download the entirety of the annotations. Most importantly we highlight the considerable amount of work that accompanies the analysis and report of biochemical data from the literature.

Nanozymes are of particular interest due to their enzyme-mimicking activity and high stability that are favorable in biomedical sensing and immunoassays. In this work, we report a highly specific N-doped nanozyme through pyrolysis of framework-confined bovine serum albumin (BSA). This strategy allows one to translate the low-cost and featureless BSA into a highly active enzyme mimic. The obtained carbon nanozyme (denoted as HBF-1-C800) displays 3- to 7-fold enhancement on peroxidase (POD) activity compared with the conventional carbon nanozymes and also shows ca. 5-fold activity enhancement compared to the reported N-doping graphene. Such excellent POD activity originates from high N-doping efficiency, protein-induced defective sites, and the intrinsic porous structure of HBF-1-C800, which provides abundantly accessible active sites and accelerates substrate diffusion simultaneously. Importantly, the HBF-1-C800 nanozyme has highly specific POD activity and also enables resistance to several harsh conditions that should denature natural enzymes. These features allow it with high accuracy, stability, and sensitivity for biosensing applications. Moreover, HBF-1-C800 has been designed as a promising platform for colorimetric biosensing of several biomarkers including H2O2, glutathione, and glucose, with wide linear ranges and low limits of detection that are satisfied with the disease diagnosis.

It has been shown that the Aspergillus flavus O-1 micromycete secretes a complex of at least two proteolytic enzymes. The most active enzyme was isolated via preparative isoelectric focusing and characterized as a serine proteinase with a molecular weight of 17 kDa and a pI of 7.82. The proteinase had a broad substrate specificity and was able to degrade fibrillar proteins such as collagen, elastin, and fibrin.

In animal feeding enzyme preparations are mainly used, which are not produced in the body of animals, although in chickens in the first weeks of life and piglets after weaning an insufficient amount of their own proteases and amylases is formed, so their inclusion in feed is also useful. By now highly active enzyme preparations with various specific activities have been created and are being produced. In feeds prepared using traditional raw materials up to 30% of nutrients are not available for digestion by enzymes secreted in the gastrointestinal tract. The inclusion of exogenous enzymes in the feed provides additional use of the indigestible fraction. It is impossible to achieve complete digestibility of feed, although in vitro exogenous enzymes digest almost 100% of the target substrates. The problem is due to the fact that natural feed does not contain pure substrates — they are associated with other substances that limit the availability of enzymes to them. The properties of natural substrates, their concentration and availability even in the same raw material are variable, which excludes reliable prediction of the effectiveness of the action of feed enzymes. Partially these difficulties are overcomed by the use of polyenzyme drugs. It is necessary to further study the specificity of native substrates and factors that increase their availability for the action of enzymes.

This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.

One of the stumbling blocks to advance the field of glycobiology has been the difficulty in synthesis of bespoke carbohydrate-based molecules like glycopolymers (e.g. human milk oligosaccharides) and glycoconjugates (e.g. glycosylated monoclonal antibodies). Recent strides towards using engineered Carbohydrate-Active enZymes (CAZymes) like glycosyl transferases, transglycosidases, and glycosynthases for glycans synthesis has allowed production of diverse glycans. Here, we discuss enzymatic routes for glycans biosynthesis and recent advances in protein engineering strategies that enable improvement of CAZyme specificity and catalytic turnover. We focus on rational and directed evolution methods that have been developed to engineer CAZymes. Finally, we discuss how improved CAZymes have been used in recent years to remodel and synthesize glycans for biotherapeutics and biotechnology related applications.

Resumen: El objetivo de esta investigación fue optimizar la inmovilización de naringinasa de Penicillium decumbens mediante atrapamiento en criogeles de alcohol polivinílico y polietilenglicol. En primer lugar, se determinó el efecto de las características de los componentes de la matriz sobre la eficacia de inmovilización y la estabilidad mecánica de los biocatalizadores obtenidos. Se empleó la herramienta de diseño de experimentos y superficie de respuesta para analizar el efecto del pH (3,0-8,0), de las concentraciones de alcohol polivinílico (5,3-8,7%) y polietilenglicol (7,0-12,0%) sobre la eficacia de inmovilización, y para optimizar estos parámetros. Adicionalmente, se estudió la capacidad de reutilización de los inmovilizados en zumos sintéticos. Se consiguieron biocatalizadores inmovilizados que conservaban un 45-47% de la enzima activa y que, tras tres ciclos de reutilización en zumo sintético, mantenían un 54,3% de la actividad inicial. Los resultados obtenidos mostraron que las condiciones de inmovilización afectaban a la eficacia de inmovilización y a la estabilidad operativa de la naringinasa atrapada.

Carbohydrates are an essential class of biomolecule and carbohydrate active enzymes (CAZys) catalyse their synthesis, refinement, and degradation, hence contributing an overall regulatory capacity to their underpinning physiological roles. As such, there is a considerable current requirement to be able to monitor, quantify and inhibit CAZy activity. It is here that Forster resonance energy transfer (FRET) is emerging as a powerful tool in enabling this, through synthetic conjugation of appropriate fluorogenic partners into a particular CAZy substrate. In this review we survey recent accomplishments in synthetic methodology for accessing defined carbohydrate structures, suitably equipped with FRET probe capability, followed by their utilisation in studying particular classes of CAZy. The majority of examples concern endo-acting glycosidases, but emergent probes for exo-glycosidases, glycosyl transferases and catalytic antibodies are also examined.

Carbohydrates are an essential class of biomolecule and carbohydrate active enzymes (CAZys) catalyze their synthesis, refinement, and degradation, hence contributing an overall regulatory capacity to their underpinning physiological roles. Here we survey recent accomplishments for accessing defined carbohydrate structures, suitably equipped with FRET probe capability, followed by their utilization in studying particular classes of CAZy.

Butyrivibrio and Pseudobutyrivibrio dominate in anaerobic gastrointestinal microbiomes, particularly the rumen, where they play a key role in harvesting dietary energy. Within these genera, five rumen species have been classified ( Butyrivibrio fibrisolvens , Butyrivibrio hungatei , Butyrivibrio proteoclasticus , Pseudobutyrivibrio ruminis and Pseudobutyrivibrio xylanivorans ) and more recently an additional Butyrivibrio sp. group was added. Given the recent increase in available genomes, we re-investigated the phylogenetic systematics and evolution of Butyrivibrio and Pseudobutyrivibrio . Across 71 genomes, we show using 16S rDNA and 40 gene marker phylogenetic trees that the current six species designations ( P. ruminis , P. xylanivorans , B. fibrisolvens , Butyrivibrio sp., B. hungatei and B. proteclasticus) are found. However, pangenome analysis showed vast genomic variation and a high abundance of accessory genes (91.50–99.34 %), compared with core genes (0.66–8.50 %), within these six taxonomic groups, suggesting incorrectly assigned taxonomy. Subsequent pangenome accessory genomes under varying core gene cut-offs (%) and average nucleotide identity (ANI) analysis suggest the existence of 42 species within 32 genera. Pangenome analysis of those that still group within B. fibrisolvens , B. hungatei and P. ruminis , based on revised ANI phylogeny, also showed possession of very open genomes, illustrating the diversity that exists even within these groups. All strains of both Butyrivibrio and Pseudobutyrivibrio also shared a broad range of clusters of orthologous genes (COGs) (870), indicating recent evolution from a common ancestor. We also demonstrate that the carbohydrate-active enzymes (CAZymes) predominantly belong to glycosyl hydrolase (GH)2, 3, 5, 13 and 43, with numerous within family isoforms apparent, likely facilitating metabolic plasticity and resilience under dietary perturbations. This study provides a major advancement in our functional and evolutionary understanding of these important anaerobic bacteria.

Stimulated drug releases in response to human movements are highly appealing in medical therapy and various daily uses. However, the design of a mechanically responsive substrate that presents high delivery capacities and can also preserve the activities of sensitive molecules such as enzymes is still challenging. Taking advantage of the recent development in effective piezoelectric flexible films and in molecular delivery devices, we propose a composite delivery substrate that preserves enzyme activities and enhances molecular delivery in response to human movements such as finger presses or massages. The substrate is achieved by combining two parts, which are the energy converting unit and the molecular loading and releasing unit. The energy converting unit is a piezoelectric-dielectric flexible composite film that produces enhanced electricity and preserves the electricity longer compared to a pure piezoelectric polymer. The molecular delivery unit is a layer-by-layer multilayer containing mesoporous silica particles that are assembled at pH 9 but used in neutral solutions. The releases of molecules including small molecules, peptides, and proteins are all accelerated in response to finger presses irrespective of the signs or densities of their charges. More importantly, the enzyme CAT preserves its activity after release from the composite substrates, meaning that the CAT-loaded (PAH/MS)n(PAH/DAS)n@rGO-TFB/PVDF-HFP composite substrate holds promise as a self-powered soothing pad that effectively removes residue H2O2.

Production of fine chemicals from biomass, in particular lignin fraction, is paramount to the development of a sustainable bioeconomy. Herein, a photosystem capable of generating H2O2 in situ from the photoreduction of O2 is presented, which is subsequently used to activate lignin peroxidase enzyme. The active enzyme selectively converts veratryl alcohol into veratryl aldehyde, a high-value flavoring and fragrance compound. This unique reaction scheme increased significantly the process greenness and safety credentials.

Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions. ispartof: MOLECULES vol:25 issue:24 ispartof: location:Switzerland status: published

Background: The enzymatic activity of the microbiome toward carbohydrates in the human digestive system is of enormous health significance (Zou, Y., et al., 2019; Pinard, D., et al., 2015). Predicting how carbohydrates through food intake may affect the distribution and balance of gut microbiota remains a major challenge. Understanding the enzyme-substrate specificity relationship of the carbohydrate-active enzyme (CAZyme) encoded by the vast gut microbiome will be an important step to address this question. In this study, we seek to establish an in-silico approach to studying the enzyme-substrate binding interaction. Results: We focused on the key carbohydrate-active enzyme (CAZyme) and established a novel Poisson noise-based few-shots learning neural network (pFSLNN) for predicting the binding affinity of indigestible carbohydrates. This approach achieved higher accuracy than other classic FSLNNs, and we have also formulated new algorithms for feature generation using only a few amino acid sequences. Sliding bin regression is integrated with mRMR for feature selection. Conclusion: The resulting pFSLNN is an efficient model to predict the binding affinity between CAZyme and common oligosaccharides. This model can be potentially applied to binding affinity prediction of other protein-ligand interactions based on limited amino acid sequences.

Penicillium subrubescens is able to degrade a broad range of plant biomass and it has an expanded set of Carbohydrate Active enzyme (CAZyme)-encoding genes in comparison to other Penicillium species. Here we used exoproteome and transcriptome analysis to demonstrate the versatile plant biomass degradation mechanism by P. subrubescens during growth on wheat bran and sugar beet pulp. On wheat bran P. subrubescens degraded xylan main chain and side residues from the Day 2 of cultivation, whereas it started to degrade side chain of pectin in sugar beet pulp prior to attacking the main chain on Day 3. In addition, on Day 3 the cellulolytic enzymes were highly increased. Our results confirm that P. subrubescens adapts its enzyme production to the available plant biomass and is a promising new fungal cell factory for the production of CAZymes.

Many insects spin cocoons to protect the pupae from unfavorable environments and predators. After emerging from the pupa, the moths must escape from the sealed cocoons. Previous works identified cocoonase as the active enzyme loosening the cocoon to form an escape-hatch. Here, using bioinformatics tools, we show that cocoonase is specific to Lepidoptera and that it probably existed before the occurrence of lepidopteran insects spinning cocoons. Despite differences in cocooning behavior, we further show that cocoonase evolved by purification selection in Lepidoptera and that the selection is more intense in lepidopteran insects spinning sealed cocoons. Experimentally, we applied gene editing techniques to the silkworm Bombyx mori, which spins a dense and sealed cocoon, as a model of lepidopteran insects spinning sealed cocoons. We knocked out cocoonase using the CRISPR/Cas9 system. The adults of homozygous knock-out mutants were completely formed and viable but stayed trapped and died naturally in the cocoon. This is the first experimental and phenotypic evidence that cocoonase is the determining factor for breaking the cocoon. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons., Author summary Spinning and cocooning are the instincts of many insects, providing a shelter to the residing pupae to resist adverse factors. After the metamorphosis of pupa into adult, the adult must break open the cocoon to emerge, which is called decocooning. We have bioinformatically identified that cocoonase is specific to Lepidoptera, and demonstrated that it is the determining factor for breaking the sealed cocoon experimentally for the first time. This work led to a novel silkworm strain yielding permanently intact cocoons and provides a new strategy for controlling the pests that form cocoons and for breeding.

The traditional classification of fungal and oomycete phytopathogens into three classes – biotrophs, hemibiotrophs, or necrotrophs – is unsustainable. This study highlights multiple phytopathogen species for which these labels have been inappropriately applied. We propose a novel and reproducible classification based solely on genome-derived analysis of carbohydrate-active enzyme (CAZyme) gene content called CAZyme-Assisted Training And Sorting of -trophy (CATAStrophy). CATAStrophy defines four major divisions for species associated with living plants. These are monomertrophs (Mo) (corresponding to biotrophs), polymertrophs (P) (corresponding to necrotrophs), mesotrophs (Me) (corresponding to hemibiotrophs), and vasculartrophs (including species commonly described as wilts, rots, or anthracnoses). The Mo class encompasses symbiont, haustorial, and non-haustorial species. Me are divided into the subclasses intracellular and extracellular Me, and the P into broad and narrow host sub-classes. This gives a total of seven discrete plant-pathogenic classes. The classification provides insight into the properties of these species and offers a facile route to develop control measures for newly recognized diseases. Software for CATAStrophy is available online at https://github.com/ccdmb/catastrophy. We present the CATAStrophy method for the prediction of trophic phenotypes based on CAZyme gene content, as a complementary method to the traditional tripartite “biotroph–hemibiotroph–necrotroph” classifications that may encourage renewed investigation and revision within the fungal biology community.

Many industrial processes may quickly proceed with biotechnological and enzymatic support. Functional and production facilitator properties of enzymes have provided variable advantages for industry. Therefore, enzyme utilization has become inevitable for industry and supplied financial advantages in case of the high energy requirement process. In the study, the amylase enzymes were isolated from thermal spring sources, and the effect on bread quality was examined. Firstly, fungal amylases were isolated from thermal spring sources (29 98 degrees C) in various places around Turkey. After determining the functional properties of amylase enzymes, the most active enzyme was used in bread production to examine their effect on bread quality. The maximum alpha-amylase activity (38.6 U/mg) has been detected in Aspergillus niger G 2-1 isolate. When compared with the commercially available one, native alpha-amylase increased the bread volume. A significant difference (p < 0.01) was found in the color properties and size of the bread between microbial produced in this study and commercially produced alpha-amylase. Five ppm alpha-amylase addition showed the optimum bread properties for dough processing.

The commercial importance of brown seaweed has been increasing over the past decade, especially due to industries interested in the extraction of phycocolloids and, more recently, of polyphenol com ...

The initial stage of in vitro polyhydroxyalkanoate (PHA) polymerization by PHA synthase from Ralstonia eutropha (PhaCRe) on a mica substrate in water was observed using high-speed scanning atomic force microscopy (HS-AFM). Before PHA polymerization, the adsorption–desorption cycle of the PhaCRe molecule on mica was observed in real time. For approximately 30 s after the addition of the PHA monomer, no significant change was observed on the mica substrate, but PhaCRe could be transformed into an active enzyme in water upon contact with the monomer during this period. Subsequently, linearly elongating rod-shaped objects were observed on the mica substrate, plausibly as a result of the polymerization reaction. The height of these elongating objects was considerably larger than the expected height for a single PHA chain. This observation suggests that PHA chains generated during the reported experiments might form some kind of a semiregular structure.

Numerous zinc ectoenzymes are folded and activated in the compartments of the early secretory pathway, such as the ER and the Golgi apparatus, before reaching their final destination. During this process, zinc must be incorporated into the active site; therefore, metalation of the nascent protein is indispensable for the expression of the active enzyme. However, to date, the molecular mechanism underlying this process has been poorly investigated. This is in sharp contrast to the physiological and pathophysiological roles of zinc ectoenzymes, which have been extensively investigated over the past decades. This manuscript concisely outlines the present understanding of zinc ectoenzyme activation through metalation by zinc and compares this with copper ectoenzyme activation, in which elaborate copper metalation mechanisms are known. Moreover, based on the comparison, several hypotheses are discussed. Approximately 80 years have passed since the first zinc enzyme was identified; therefore, it is necessary to improve our understanding of zinc ectoenzymes from a biochemical perspective, which will further our understanding of their biological roles.

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

The protective immunological effects of the influenza neuraminidase (NA) surface protein are of renewed interest but NA content in vaccines remains unstandardized and methods to easily and reliably quantify NA content are unsatisfactory. We describe the use of a recently developed fluorometric titration reagent, TR1, to efficiently quantify the total enzymatically active NA content of six commercially-available influenza vaccines, including split/subunit, inactivated/live and standard /high dose products distributed from 2015/16 to 2017/18 in North America. Considerable differences in active NA content were measured between influenza vaccine products for the same season, with relative content differences between brands generally maintained across seasons. These results highlight the simplicity of use of this reagent, and its unique ability to quantitate enzymatically active NA without the need for specific activities of individual enzymes. The reagent could also prove valuable in assessing the importance of using fully active enzyme to generate protective immune responses.

Development of protein cages for encapsulation of active enzyme cargoes and their subsequent arrangement into a controllable three-dimensional array is highly desirable. However, cargo capture is typically challenging because of difficulties in achieving reversible assembly/disassembly of protein cages in mild conditions. Herein we show that by using an unusual ferritin cage protein that undergoes triggerable assembly under mild conditions, we can achieve reversible filling with protein cargoes including an active enzyme. We demonstrate that these filled cages can be arrayed in three-dimensional crystal lattices and have an additional chaperone-like effect, increasing both thermostability and enzymatic activity of the encapsulated enzyme.

Over the past few decades, the use of enzyme preparation in food processing has increased tremendously because it is a green technology. Plants, animals, and microorganisms are important sources of enzyme preparation, containing an active enzyme or a mixture of two or more active enzymes. Alcalase, Flavourzyme, and Protamex are among the proteases intentionally added to foodstuffs as processing aids to impart flavor, taste, and textural changes in processed food. Recently, these proteases have gained the attention of the food industry for products of protein hydrolysis in other food applications, particularly in the production of bioactive peptides having physiological functions. Numerous food and food processing by-products high in protein content as the substrates for the proteases have been extensively studied. The potential of bioactive peptides in reducing the worldwide epidemic of noncommunicable diseases (NCDs) seems promising in a significant amount of the studies conducted. Tackling NCDs, which account for millions of premature deaths annually, constitutes one of the major challenges for development in modern days. The gap between laboratory experiment and industrial product realization must be filled to bring about an improvement in nutritional status and the physical well-being of people. This chapter describes proteases from fundamental to recent updates as well as discusses the feasibility and possible challenges of the industry application of proteases for the production of bioactive peptides. The potential roles of proteases in combating food insecurity and mitigating environmental issues by ensuring optimum utilization of food and food processing by-products will be highlighted.

Rational enzyme design presents a major challenge that has not been overcome by computational approaches. One of the key challenges is the difficulty in assessing the magnitude of the maximum possible catalytic activity. In an attempt to overcome this challenge, we introduce a strategy that takes an active enzyme (assuming that its activity is close to the maximum possible activity), design mutations that reduce the catalytic activity, and then try to restore that catalysis by mutating other residues. Here we take as a test case the enzyme haloalkane dehalogenase (DhlA), with a 1,2-dichloroethane substrate. We start by demonstrating our ability to reproduce the results of single mutations. Next, we design mutations that reduce the enzyme activity and finally design double mutations that are aimed at restoring the activity. Using the computational predictions as a guide, we conduct an experimental study that confirms our prediction in one case and leads to inconclusive results in another case with 1,2-dichloroethane as substrate. Interestingly, one of our predicted double mutants catalyzes dehalogenation of 1,2-dibromoethane more efficiently than the wild-type enzyme.

Gaucher disease, an autosomal recessive disorder, is caused by a deficiency of glucocerebrosidase (GCase) enzyme, a peripheral membrane-associated glycoprotein that hydrolyses glucosylceramide in lysosomes. Glycosylation is essential for the development of a catalytically active enzyme, specifically in the first site, located at Asn19. However, both the molecular basis of the relevance of N-glycosylation over GCase activity and the effects of glycosylation over its structure and dynamics are still not fully understood. Thus, the present work evaluated GCase enzyme in increasing glycosylation content using triplicate unbiased molecular dynamics simulations. Accordingly, the N-linked glycan chains caused local conformational stabilization effects over the protein, as well as in regions flanking the enzyme catalytic dyad. In the case of the Asn19-linked glycan, it also occurred around region 438-444, where one of the most prevalent GCase mutations is found. Markedly, an increasing catalytic dyad organization was related to increasing glycosylation contents, offering the first atomic-level explanation for the experimental observation that GCase activity is controlled by glycosylation, especially at Asn19.

Proteases have been important diagnostic indicators because of their relevance to disease states. Therefore, different types of assay methods have been developed for the accurate measurement of protease activity. In this study, we report a colorimetric protease activity assay method for matrix metalloproteinase 2 (MMP2) using an engineered procaspase-3. The tailor-made procaspase-3 is activated by MMP2, and the active enzyme produces a yellow absorbance signal by hydrolyzing a colorimetric caspase-3 substrate. Optimization of this assay system permitted the assessment of MMP2 activity at a picomolar concentration range even though the assay system is based on absorbance. The enzymatic reaction cascade leads to signal amplification and contributes to the high sensitivity of the assay method. The sensitive and simple assay method developed in this study can be developed as a diagnostic tool for MMP2-associated diseases. Additionally, the design strategy of procaspase-3 can be generalized, and the technology platform is expected to be easily adapted for other proteases.

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor.

Summary Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory “B” subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 A structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

Raman spectroscopy is used to study components of an enzyme-catalyzed reaction. Time dependences of integral Raman intensities in the spectra of reaction mixtures are analyzed. Parameters of chymotrypsin-catalyzed reaction are determined, and the concentrations of active enzyme molecules in the samples from different manufacturers are compared.

To study factors that affect WPD-loop motion in protein tyrosine phosphatases (PTPs), a chimera of PTP1B and YopH was created by transposing the WPD loop from PTP1B to YopH. Several subsequent mutations proved to be necessary to obtain a soluble, active enzyme. That chimera, termed chimera 3, retains productive WPD-loop motions and general acid catalysis with a pH dependency similar to that of the native enzymes. Kinetic isotope effects show the mechanism and transition state for phosphoryl transfer are unaltered. Catalysis of the chimera is slower than that of either of its parent enzymes, although its rate is comparable to those of most native PTPs. X-ray crystallography and nuclear magnetic resonance were used to probe the structure and dynamics of chimera 3. The chimera's structure was found to sample an unproductive hyper-open conformation of its WPD loop, a geometry that has not been observed in either of the parents or in other native PTPs. The reduced catalytic rate is attributed to the protein's sampling of this conformation in solution, reducing the fraction in the catalytically productive loop-closed conformation.

We report that long double-stranded DNA confined to quasi-1D nanochannels undergoes superdiffusive motion under the action of the enzyme T4 DNA ligase in the presence of necessary co-factors. Inside the confined environment of the nanochannel, double-stranded DNA molecules stretch out due to self-avoiding interactions. In absence of a catalytically active enzyme, we see classical diffusion of the center of mass. However, cooperative interactions of proteins with the DNA can lead to directed motion of DNA molecules inside the nanochannel. Here we show directed motion in this configuration for three different proteins (T4 DNA ligase, MutS, E. coli DNA ligase) in the presence of their energetic co-factors (ATP, NAD+).

O-Ureidoserine racemase (DcsC) is a PLP-independent enzyme in the biosynthetic route to the antibiotic d-cycloserine. Here we present the recombinant expression and characterization of a significantly more active DcsC variant featuring an N-terminal SUMO-tag. Synthesis of enantiomeric pure inhibitors in combination with site-specific mutation of active site cysteines to serines of this enzyme offers closer insights into the mechanism of this transformation. Homology modelling with a close relative (diaminopimelate epimerase, DapF) inspired C- and N-terminal truncation of DcsC to produce a more compact yet still active enzyme variant.

Glycosidases mediate the fragmentation of glycoconjugates in the body, including the vital recycling of endogenous molecules. Several inherited diseases in man concern deficiencies in lysosomal glycosidases degrading glycosphingolipids. Prominent is Gaucher disease caused by an impaired lysosomal β-glucosidase (glucocerebrosidase, GBA) and resulting in pathological lysosomal storage of glucosylceramide (glucocerebroside) in tissue macrophages. GBA is a retaining glucosidase with a characteristic glycosyl–enzyme intermediate formed during catalysis. Using the natural suicide inhibitor cyclophellitol as a lead, we developed mechanism-based irreversible inhibitors of GBA equipped with a fluorescent reporter. These reagents covalently link to the catalytic nucleophile residue of GBA and permit specific and sensitive visualization of active enzyme molecules. The amphiphilic activity-based probes (ABPs) allow in situ detection of active GBA in cells and organisms. Furthermore, they may be used to biochemically confirm the diagnosis of Gaucher disease and they might assist in screening for small compounds interacting with the catalytic pocket. While the focus of this chapter is ABPs for β-glucosidases and Gaucher disease, the described concept has meanwhile been extended to other retaining glycosidases and related disease conditions as well.