Enzyme Specificity Research Articles

Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging.

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.

Specificity of Rhodococcus opacus 1CP cells' responses to benzoate and 3-chlorobenzoate.

Halogenated aromatic compounds are more resistant to microbial degradation than non-halogenated aromatic compounds. Microbial degradation of sodium benzoate in the presence of sodium 3-chlorobenzoate is of interest. The ability to degrade aromatic compounds is largely determined by the substrate specificity of the first enzyme that initiates degradation, namely, benzoate 1,2-dioxygenase for benzoate degradation, and 3-chlorobenzoate 1,2-dioxygenase for 3-chlorobenzoate degradation. In this study, the perspective of immobilized cells of Rhodococcus opacus 1CP actinobacterium for degradation of benzoate and 3-chlorobenzoate was explored. The biosensor approach (a membrane microbial sensor based on immobilized cells of Rhodococcus opacus 1CP and the Clark-type oxygen electrode as a transducer) was applied to evaluate the actinobacterial cells' responses to benzoate and 3-chlorobenzoate in the absence of both enzymes, benzoate 1,2-dioxygenase and 3-chlorobenzoate 1,2-dioxygenase, or in the presence of one of the said enzymes. Data obtained show that 1CP actinobacterium possessed a constitutive system for the transport of benzoate and 3-chlorobenzoate into culture cells. The affinity of the transport system for benzoate was higher than that for 3-chlorobenzoate. Moreover, adaptation to one substrate did not preclude the use of the second substrate. Probably, porins facilitated the penetration of benzoate and 3-chlorobenzoate into 1CP cells. Analyzing V vs. S dependencies, negative cooperativity was found, when benzoate 1,2-dioxygenase bound substrate (3-chlorobenzoate), while positive cooperativity was determined at benzoate binding. The observed difference could be associated with the presence of at least two systems of 3-chlorobenzoate transport into actinobacterial cells and allosteric interaction of active sites of benzoate 1,2-dioxygenase in the presence of 3-chlorobenzoate. The membrane microbial sensor based on immobilized Rhodococcus opacus 1CP cells could be useful as a perspective tool for comparative evaluation of enzymes of complex structure such as benzoate- and 3-chlorobenzoate 1,2-dioxygenase.

Multipoint Immobilization at the Inert Center of Urease on Homofunctional Diazo-Activated Silica Gel: A Way of Restoring Room-Temperature Catalytic Sustainability for Perennial Utilization.

At present, enzyme immobilization is a big issue. It improves enzyme stability, activity, specificity, or selectivity, particularly the enantioselectivity compared to the native enzymes, and by solving the separation problem, it helps in recovering the catalyst with good reusability as desired in vitro. Motivated by these facts, in this work, Jack bean urease (JBU) is immobilized on three-dimensional (3D)-network silica gel (SG) via multipoint covalent bonding employing dimethyldichlorosilane (DMDCS) and p-nitrophenol, respectively, as the second-generation silane-coupling reagent and spacer. The homofunctional diazo group appearing at the functionalized SG unit cell makes a diazo linkage at the inert center, the ortho position of the phenolic-OH of the tyrosine moiety, where all of the amino, thiol, phenol, imidazole, carboxy, etc., groups of the enzyme residues, including those that belong to the active site, remain intact. The coupling process, analyzed using field emission scanning electron microscopy (FESEM), energy-dispersive X-ray analysis (EDX), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet-visible spectroscopy (UV-vis), and fluorescence spectroscopy, occurs without molecular aggregation in borate buffer at pH 8.8 ± 0.4, which is much higher than the iso-electric point (pH 5.1) of the macromolecule where it becomes soluble. Eventually, the immobilization is maximize and also the native-enzyme activities are restored remarkably. The immobilized catalyst converts urea (0.0625-0.15 mmol L-1) to ammonia appreciably (94.50 ± 1.5%) at 27 °C, and the efficiency is well comparable to that of the native enzyme (93.0 ± 0.4%). The efficiency gradually diminishes, coming down to 50% at the 40th cycle, and the enzyme returns to its native conformation within 72 h in tris-EDTA borate buffer at 27 °C for the next 40 cycles of reuse and so on. The efficiency becomes hindered by 8-10% in every 5th subsequent reuse to reach 50% on the 30th reuse, resulting in room-temperature catalytic sustainability of 90 days. The catalytic performances are well restored in rice extract and coconut water.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic β-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23–31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

Identification and structural prediction of the unrevealed amidohydrolase enzyme: Pterin deaminase from Agrobacterium tumefaciens LBA4404.

Microbes make a remarkable contribution to the health and well-being of living beings all over the world. Interestingly, pterin deaminase is an amidohydrolase enzyme that exhibits antitumor, anticancer activities and antioxidant properties. With the existing evidence of the presence of pterin deaminase from microbial sources, an attempt was made to reveal the existence of this enzyme in the unexplored bacterium Agrobacterium tumefaciens LBA4404. After, the cells were harvested and characterized as intracellular enzymes and then partially purified through acetone precipitation. Subsequently, further purification stepwas carried out with an ion-exchange chromatogram (HiTrap Q FF) using theFast-Protein Liquid Chromatography technique (FPLC). Henceforward, the approximate molecular weight of the purified pterin deaminase was determined through SDS-PAGE. Furthermore, the purified protein was identified accurately by MALDI-TOF, and the sequence was explored through a Mascot search engine. Additionally, the three-dimensional structure was predicted and then validated, as well as ligand-binding sites, and the stability of this enzyme was confirmed for the first time. Thus, the present study revealed the selected parameters showing a considerable impact on the identification and purification of pterin deaminase from A. tumefaciens LBA4404 for the first time. The enzyme specificity makes it a favorable choice as a potent anticancer agent.

Free Radical Scavenging, Redox Balance and Wound Healing Activity of Bioactive Peptides Derived from Proteinase K-Assisted Hydrolysis of Hypophthalmichthys molitrix Skin Collagen.

Various protocols for enzymatic hydrolysis of fish by-products are increasingly tested to ensure value-added products with functional and biological properties important for food, cosmetic and medical applications. In addition, they attempt to minimize waste from industrial processing and environmental requirements. This study aims to establish an efficient protocol based on two-step enzymatic hydrolysis of freshwater fish skin and to evaluate the effect of resulting bioactive peptides on free radical scavenging, redox balance and regulation of fibroblast proliferation and migration. Pepsin-soluble collagen extracted from silver carp (Hypophthalmichthys molitrix) skin was hydrolyzed by proteinase K at specific sites under controlled conditions. The molecular mass of ultrafiltration permeate was determined by gradient electrophoresis and gel filtration chromatography. The biological activity of intermediate and small size bioactive peptides was evaluated in experimental models in vitro mimicking oxidative stress and skin wound conditions. Extracted fish collagen was hydrolysed using proteinase K, the most efficient enzyme for the cleavage of the primary structure of the molecule, as previously found in silico. Established optimal conditions increased the enzyme specificity and the process yield. Bioactive peptides exerted significantly higher scavenging activity on free stable radicals and hydroxyl radicals often found in vivo, compared to fish collagen. They stimulated fibroblast metabolism in a dose-dependent manner and up-regulated cell migration in a scratch wound model. Pretreatment of fibroblasts with induced oxidative stress using optimal concentrations of fish peptides prevented the increase of reactive oxygen species production. In conclusion, bioactive peptides from carp skin demonstrated valuable properties of maintaining redox balance and skin wound healing process improvement, which indicated further potential applications in the development of pharmaceutical and nutraceutical formulations. In this study the enzymatic hydrolysis was applied to isolated protein, in contrast to previous studies using waste tissue with variable composition. Recovered bioactive peptides acted not only as antioxidant agents, but also as regulators of oxidative stress and wound healing processes in skin cell models. Their nutritional and cosmetic application is recommended in novel formulations fighting skin ageing phenomena.

Adaptation of thermophilic enzymes from an ancestral reductive TCA cycle for carbon fixation in plants

The reductive TCA cycle (rTCA) is one of 5 carbon‐fixing cycles in prokaryotes other than the Calvin‐Benson cycle ubiquitous in plants. Adaptation of these cycles to biofuel crops is one strategy for reducing the greenhouse gas CO2 and simultaneously increasing crop yields. A minimal 5‐enzyme synthetic rTCA cycle was designed at NCSU utilizing 2 unique enzymes to carboxylate and reduce 2‐oxoglutarate to isocitrate. The enzymes are derived from an evolutionarily primitive configuration of the rTCA cycle that persists in the order Aquificales in hot springs and deep ocean thermal vents. Two strategies are being pursued to engineer mesophilic forms of these enzymes: 1) reducing the temperature of activity of the Aquificales enzymes, and 2) modifying the substrate specificity of homologous mesophilic enzymes. The first enzyme, 2‐oxoglutarate carboxylase (OGC), is a member of the biotin‐dependent carboxylase family homologous to pyruvate carboxylase. Biotin‐dependent carboxylases are multi‐subunit complexes. So far, we have identified the temperature‐dependent steps of the biotin carboxylase domain of OGC and predicted mutations to reduce the temperature of activity based on the crystal structure. Phylogenetic analysis suggests that OGC and pyruvate carboxylase diverged prior to Aquificales evolution. An ancestral promiscuous enzyme has been proposed to carboxylate both pyruvate and 2‐oxoglutarate. Ancestral enzymes are being reconstructed from phylogenetic analyses to switch the substrate specificity of mesophilic pyruvate carboxylase to carboxylate 2‐oxoglutarate. The second enzyme, oxalosuccinate reductase (OSR), is homologous with isocitrate dehydrogenase. In fact, isocitrate dehydrogenase has replaced OGC/OSR in most organisms utilizing the rTCA cycle. Isocitrate dehydrogenase favors the oxidative decarboxylase of isocitrate in the TCA cycle, while OGC/OSR favors the reductive cycle. OSR is an ancestral, non‐decarboxylating isocitrate dehydrogenase. We propose to engineer a mesophilic OSR by mutating isocitrate dehydrogenase to inhibit decarboxylation, based on phylogenic and structural studies of OSR. Ultimately engineering a mesophilic OGC/OSR will be utilized to increase carbon capture in seed oil crops like Camelina sativa that are carbon limited. Oil from Camelina seeds can be converted to jet fuels.

Coupling auxin‐inducible degradation with quantitative proteomics for in‐vivoprotein functional characterization

Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting from the permanent or long‐term changes to the target protein expression/function. In contrast, the auxin‐inducible degradation (AID) system allows very rapid depletion of endogenous target protein levels, thereby minimizing deleterious effects from long‐term perturbation caused by some traditional methodologies. Combining the AID system with quantitative proteomics in applicable model systems allows detection of proteome changes immediately following target protein degradation. The rapid depletion achieved by AID is well‐suited to studying the biological specificity of regulatory enzymes and confidently identifying their direct substrates. Our lab is interested in understanding the role of phosphatases in the regulation of the cell cycle. We demonstrate the power of combining AID and proteomics in the budding yeast, S. cerevisiae, by targeting components of protein phosphatase 2A (PP2A), an abundant heterotrimeric phosphatase with numerous substrates involved in multiple cellular processes, including the cell cycle. The AID system achieves ≥ 85% reduction in the abundance of PP2A components within 10‐15 minutes after auxin addition, resulting in detectable phosphoproteome changes by 20 minutes. The rapid response time allows us to selectively probe the proximate phosphoproteome changes enriched with direct PP2A substrates while minimizing long‐term, indirect effects resulting from slower transcriptional changes and cellular adaptation responses. Current work is focused on applying this system to studying the contributions of different PP2A complexes at different cell cycle stages, providing novel information on PP2A regulatory contributions to this fundamental biological process. The AID system coupled with proteomics is broadly applicable for studying the role of almost any protein in diverse physiological settings and model organisms.

Kinase Domain Autophosphorylation Rewires the Activity and Substrate Specificity of CK1 Enzymes

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N‐terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation‐induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high‐affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

Convergent gene clusters underpin hyperforin biosynthesis in St John's wort.

The meroterpenoid hyperforin is responsible for the antidepressant activity of St John's wort extracts, but the genes controlling its biosynthesis are unknown. Using genome mining and biochemical work, we characterize two biosynthetic gene clusters (BGCs) that encode the first three steps in the biosynthesis of hyperforin precursors. The findings of syntenic and phylogenetic analyses reveal the parallel assembly of the two BGCs. The syntenous BGC in Mesua ferrea indicates that the first cluster was assembled before the divergence of the Hypericaceae and Calophyllaceae families. The assembly of the second cluster is the result of a coalescence of genomic fragments after a major duplication event. The differences between the two BGCs - in terms of gene expression, response to methyl jasmonate, substrate specificity and subcellular localization of key enzymes - suggest that the presence of the two clusters could serve to generate separate pools of precursors. The parallel assembly of two BGCs with similar compositions in a single plant species is uncommon, and our work provides insights into how and when these gene clusters form. Our discovery helps to advance our understanding of the evolution of plant specialized metabolism and its genomic organization. Additionally, our results offer a foundation from which hyperforin biosynthesis can be more fully understood, and which can be used in future metabolic engineering applications.

Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides

BackgroundNon-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides.ResultsHere, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation.ConclusionThis study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.

LoopGrafter: a web tool for transplanting dynamical loops for protein engineering.

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

Allosteric regulation by membranes and hydrophobic subsites in phospholipase A2 enzymes determine their substrate specificity

Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including obesity, insulin sensitivity, and cardiovascular diseases. However, defining the substrate specificity of enzymes of lipid metabolism is a challenging task. For example, phospholipase A2 (PLA2) enzymes constitute a superfamily of degradative, biosynthetic, and signaling enzymes that all act stereospecifically to hydrolyze and release the fatty acids of membrane phospholipids. This review focuses on how membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation and other diseases. Our group has developed “substrate lipidomics” to quantify the substrate phospholipid specificity of each PLA2 and coupled this with molecular dynamics simulations to reveal that enzyme specificity is linked to specific hydrophobic binding subsites for membrane phospholipid substrates. We have also defined unexpected headgroup and acyl chain specificity for each of the major human PLA2 enzymes, which explains the observed specificity at a structural level. Finally, we discovered that a unique hydrophobic binding site—and not each enzyme’s catalytic residues or polar headgroup binding site—predominantly determines enzyme specificity. We also discuss how PLA2s release specific fatty acids after allosteric enzyme association with membranes and extraction of the phospholipid substrate, which can be blocked by stereospecific inhibitors. After decades of work, we can now correlate PLA2 specificity and inhibition potency with molecular structure and physiological function.

Structure, Function and Protein Engineering of Cereal-Type Inhibitors Acting on Amylolytic Enzymes.

Numerous plants, including cereals, contain seed proteins able to inhibit amylolytic enzymes. Some of these inhibitors, the CM-proteins (soluble in chloroform:methanol mixtures)—also referred to as cereal-type inhibitors (CTIs)—are the topic of this review. CM-proteins were first reported 75 years ago. They are small sulfur-rich proteins of the prolamine superfamily embracing bifunctional α-amylase/trypsin inhibitors (ATIs), α-amylase inhibitors (AIs), limit dextrinase inhibitors (LDIs), and serine protease inhibitors. Phylogenetically CM-proteins are predicted across poaceae genomes and many isoforms are identified in seed proteomes. Their allergenicity and hence adverse effect on humans were recognized early on, as were their roles in plant defense. Generally, CTIs target exogenous digestive enzymes from insects and mammals. Notably, by contrast LDI regulates activity of the endogenous starch debranching enzyme, limit dextrinase, during cereal seed germination. CM-proteins are four-helix bundle proteins and form enzyme complexes adopting extraordinarily versatile binding modes involving the N-terminal and different loop regions. A number of these inhibitors have been characterized in detail and here focus will be on target enzyme specificity, molecular recognition, forces and mechanisms of binding as well as on three-dimensional structures of CM-protein–enzyme complexes. Lastly, prospects for CM-protein exploitation, rational engineering and biotechnological applications will be discussed.

In silico evaluation of a targeted metaproteomics strategy for broad screening of cellulolytic enzyme capacities in anaerobic microbiome bioreactors

BackgroundMicrobial-driven solubilization of lignocellulosic material is a natural mechanism that is exploited in anaerobic digesters (ADs) to produce biogas and other valuable bioproducts. Glycoside hydrolases (GHs) are the main enzymes that bacterial and archaeal populations use to break down complex polysaccharides in these reactors. Methodologies for rapidly screening the physical presence and types of GHs can provide information about their functional activities as well as the taxonomical diversity within AD systems but are largely unavailable. Targeted proteomic methods could potentially be used to provide snapshots of the GHs expressed by microbial consortia in ADs, giving valuable insights into the functional lignocellulolytic degradation diversity of a community. Such observations would be essential to evaluate the hydrolytic performance of a reactor or potential issues with it.ResultsAs a proof of concept, we performed an in silico selection and evaluation of groups of tryptic peptides from five important GH families derived from a dataset of 1401 metagenome-assembled genomes (MAGs) in anaerobic digesters. Following empirical rules of peptide-based targeted proteomics, we selected groups of shared peptides among proteins within a GH family while at the same time being unique compared to all other background proteins. In particular, we were able to identify a tractable unique set of peptides that were sufficient to monitor the range of GH families. While a few thousand peptides would be needed for comprehensive characterization of the main GH families, we found that at least 50% of the proteins in these families (such as the key families) could be tracked with only 200 peptides. The unique peptides selected for groups of GHs were found to be sufficient for distinguishing enzyme specificity or microbial taxonomy. These in silico results demonstrate the presence of specific unique GH peptides even in a highly diverse and complex microbiome and reveal the potential for development of targeted metaproteomic approaches in ADs or lignocellulolytic microbiomes. Such an approach could be valuable for estimating molecular-level enzymatic capabilities and responses of microbial communities to different substrates or conditions, which is a critical need in either building or utilizing constructed communities or defined cultures for bio-production.ConclusionsThis in silico study demonstrates the peptide selection strategy for quantifying relevant groups of GH proteins in a complex anaerobic microbiome and encourages the development of targeted metaproteomic approaches in fermenters. The results revealed that targeted metaproteomics could be a feasible approach for the screening of cellulolytic enzyme capacities for a range of anaerobic microbiome fermenters and thus could assist in bioreactor evaluation and optimization.

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The kcat/Km values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.

The Role of Lysine Methyltransferase SET7/9 in Proliferation and Cell Stress Response.

Lysine-specific methyltransferase 7 (KMT7) SET7/9, aka Set7, Set9, or SetD7, or KMT5 was discovered 20 years ago, yet its biological role remains rather enigmatic. In this review, we analyze the particularities of SET7/9 enzymatic activity and substrate specificity with respect to its biological importance, mostly focusing on its two well-characterized biological functions: cellular proliferation and stress response.

Physcomitrium (Physcomitrella) patens endo-glucanase 16 is involved in the cell wall development of young tissue.

Plants maintain large repertoires of carbohydrate-active enzymes (CAZymes)-comprising between 3% and 10% of their genomes-to synthesize, modify, and degrade the polysaccharide components of the cell wall. We recently identified a unique group of plant endo-glucanases from Glycoside Hydrolase Family 16, viz. EG16 orthologs, which constitute a sister clade to the well-known XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products. Biochemical analysis of EG16 orthologs from poplar (Populus trichocarpa), grapevine (Vitis vinifera), and spreading earthmoss (Physcomitrium patens) has demonstrated that these endo-glucanases are distinctly active on cell wall matrix glycans, mixed-linkage β(1,3);β(1,4)-glucan and xyloglucan (XyG), and that enzyme structure and specificity is highly conserved across diverse plant lineages. However, the physiological role of EG16 orthologs in any species is presently unknown. To shed light on EG16 function in vivo, here we performed reverse genetics and protein localization analyses of the single EG16 ortholog in the model moss P. patens, where this gene is highly expressed in young, expanding tissues, particularly in protonema. Surprisingly, deletion of the PpEG16 gene by homologous recombination led to an increase in growth, as well as accelerated senescence. Notably, the PpEG16 protein was shown to co-localize with XyG in the cell wall of protonema tissue, specifically at cell tips, despite lacking a secretion signal peptide. Although the precise biological role of EG16 orthologs remains elusive, our results implicate these highly conserved glycoside hydrolases in cell wall polysaccharide remodeling and recycling. We anticipate that these foundational results will inform future studies on EG16 function across plant lineages.