Introducing a new type of alternative laryngeal mucosa model.

Research of human vocal fold (VF) biology is hampered by several factors. The sensitive microstructure of the VF mucosa is one of them and limits the in vivo research, as biopsies carry a very high risk of scarring. A laryngeal organotypic model consisting of VF epithelial cells and VF fibroblasts (VFF) may overcome some of these limitations. In contrast to human VFF, which are available in several forms, availability of VF epithelial cells is scarce. Buccal mucosa might be a good alternative source for epithelial cells, as it is easily accessible, and biopsies heal without scarring. For this project, we thus generated alternative constructs consisting of immortalized human VF fibroblasts and primary human buccal epithelial cells. The constructs (n = 3) were compared to native laryngeal mucosa at the histological and proteomic level. The engineered constructs reassembled into a mucosa-like structure after a cultivation period of 35 days. Immunohistochemical staining confirmed a multi-layered stratified epithelium, a collagen type IV positive barrier-like structure resembling the basement membrane, and an underlying layer containing VFF. Proteomic analysis resulted in a total number of 1961 identified and quantified proteins. Of these, 83.8% were detected in both native VF and constructs, with only 53 proteins having significantly different abundance. 15.3% of detected proteins were identified in native VF mucosa only, most likely due to endothelial, immune and muscle cells within the VF samples, while 0.9% were found only in the constructs. Based on easily available cell sources, we demonstrate that our laryngeal mucosa model shares many characteristics with native VF mucosa. It provides an alternative and reproducible in vitro model and offers many research opportunities ranging from the study of VF biology to the testing of interventions (e.g. drug testing).

The Crystal Structure of Mouse Ces2c, a Potential Ortholog of Human CES2, Shows Structural Similarities in Substrate Regulation and Product Release to Human CES1.

Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/ß hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members.

Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand.

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.

Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation.

Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3' region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags.

Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.

Hepatocyte Proteome Alterations Induced by Individual and Combinations of Common Free Fatty Acids.

Non-alcoholic fatty liver disease is a pathology with a hard-to-detect onset and is estimated to be present in a quarter of the adult human population. To improve our understanding of the development of non-alcoholic fatty liver disease, we treated a human hepatoma cell line model, HepG2, with increasing concentrations of common fatty acids, namely myristic, palmitic and oleic acid. To reproduce more physiologically representative conditions, we also included combinations of these fatty acids and monitored the cellular response with an in-depth proteomics approach and imaging techniques. The two saturated fatty acids initially presented a similar phenotype of a dose-dependent decrease in growth rates and impaired lipid droplet formation. Detailed analysis revealed that the drop in the growth rates was due to delayed cell-cycle progression following myristic acid treatment, whereas palmitic acid led to cellular apoptosis. In contrast, oleic acid, as well as saturated fatty acid mixtures with oleic acid, led to a dose-dependent increase in lipid droplet volume without adverse impacts on cell growth. Comparing the effects of harmful single-fatty-acid treatments and the well-tolerated fatty acid mixes on the cellular proteome, we were able to differentiate between fatty-acid-specific cellular responses and likely common lipotoxic denominators.

Descriptive proteomics of paired human vocal fold and buccal mucosa tissue.

The vast majority of voice disorders is associated with changes of the unique, but delicate, human vocal fold mucosa. The ability to develop new effective treatment methods is significantly limited by the physical inaccessibility and the extremely rare occasions under which healthy tissue biopsies can be obtained. Therefore, the interest in laryngological research has shifted to human oral (buccal) mucosa, a similar and more easily available tissue. The harvesting process is less invasive and accompanied with faster healing and less scarring, compared to vocal fold mucosa. Here we report a descriptive proteomic comparison of paired human buccal and vocal fold mucosa by high-resolution mass spectrometry (CID-MS/MS). Our study identified a total of 1575 proteins detected within both tissues that are highly consistent in several crucial biological processes, cellular components, and molecular functions. Hence, our proteomic analysis will provide a fundamental resource for the laryngological research community.

Transcriptome and proteome of the corm, leaf and flower of Hypoxis hemerocallidea (African potato).

The corm of Hypoxis hemerocallidea, commonly known as the African potato, is used in traditional medicine to treat several medical conditions such as urinary infections, benign prostate hyperplasia, inflammatory conditions and testicular tumours. The metabolites contributing to the medicinal properties of H. hemerocallidea have been identified in several studies and, more recently, the active terpenoids of the plant were profiled. However, the biosynthetic pathways and the enzymes involved in the production of the terpene metabolites in H. hemerocallidea have not been characterised at a transcriptomic or proteomic level. In this study, total RNA extracted from the corm, leaf and flower tissues of H. hemerocallidea was sequenced on the Illumina HiSeq 2500 platform. A total of 143,549 transcripts were assembled de novo using Trinity and 107,131 transcripts were functionally annotated using the nr, GO, COG, KEGG and SWISS-PROT databases. Additionally, the proteome of the three tissues were sequenced using LC-MS/MS, revealing aspects of secondary metabolism and serving as data validation for the transcriptome. Functional annotation led to the identification of numerous terpene synthases such as nerolidol synthase, germacrene D synthase, and cycloartenol synthase amongst others. Annotations also revealed a transcript encoding the terpene synthase phytoalexin momilactone A synthase. Differential expression analysis using edgeR identified 946 transcripts differentially expressed between the three tissues and revealed that the leaf upregulates linalool synthase compared to the corm and the flower tissues. The transcriptome as well as the proteome of Hypoxis hemerocallidea presented here provide a foundation for future research.

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.

Comparative proteomics of common allergenic tree pollens of birch, alder, and hazel.

BACKGROUND: In addition to known allergens, other proteins in pollen can aid the development of an immune response in allergic individuals. The contribution of the "unknown" protein allergens is apparent in phylogenetically related species where, despite of high homology of the lead allergens, the degree of allergenic potential can vary greatly. The aim of this study was to identify other potentially allergenic proteins in pollen of three common and highly related allergenic tree species: birch (Betula pendula), hazel (Corylus avellana) and alder (Alnus glutinosa). METHODS: For that purpose, we carried out a comprehensive, comparative proteomic screening of the pollen from the three species. In order to maximize protein recovery and coverage, different protein extraction and isolation strategies during sample preparation were employed. RESULTS: As a result, we report 2500-3000 identified proteins per each of the pollen species. Identified proteins were further used for a number of annotation steps, providing insight into differential distribution of peptidases, peptidase inhibitors and other potential allergenic proteins across the three species. Moreover, we carried out functional enrichment analyses that, interestingly, corroborated high species similarity in spite of their relatively distinct protein profiles. CONCLUSION: We provide to our knowledge first insight into proteomes of two very important allergenic pollen types, hazel and alder, where not even transcriptomics data are available, and compared them to birch. Datasets from this study can be readily used as protein databases and as such serve as basis for further functional studies.

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases.

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Proteomic analysis of soluble protein extract of adult Toxocara cati.

Toxocara cati is a cat roundworm and the causative agent of toxocariasis as a cosmopolitan zoonotic disease. As no information has been reported so far, identification of T. cati proteins can be useful for the development of new diagnostic strategies. This study was conducted to identify the major proteins in the adult T. cati tegument using bi-dimensional electrophoresis (2-DE) and shotgun proteomics. A total proteins were identified, among them the metabolic enzymes were the largest group, including: Enolase, triose phosphate isomerase, fructose-bisphosphate aldolase, aldehyde dehydrogenase. The other important protein groups recognized in T. cati, belong to the HSP-family, the structure and motor proteins, such as actin. The role of these proteins have been implicated in parasite-host interactions and modulating cellular immune response, immune regulation in evasion mechanisms of the host immune response. Characterizing T. cati adult proteins play a key role not only in host-parasite interactions, but also in the discovery of drug targets, subunit vaccines against toxocariasis, immunodiagnostic kits for toxocariasis and the identification of novel immuno-modulators that can form the next generation of therapeutic possibilities for inflammatory diseases.

Folding Assessment of Incorporation of Noncanonical Amino Acids Facilitates Expansion of Functional-Group Diversity for Enzyme Engineering.

Protein design is limited by the diversity of functional groups provided by the canonical protein "building blocks". Incorporating noncanonical amino acids (ncAAs) into enzymes enables a dramatic expansion of their catalytic features. For this, quick identification of fully translated and correctly folded variants is decisive. Herein, we report the engineering of the enantioselectivity of an esterase utilizing several ncAAs. Key for the identification of active and soluble protein variants was the use of the split-GFP method, which is crucial as it allows simple determination of the expression levels of enzyme variants with ncAA incorporations by fluorescence. Several identified variants led to improved enantioselectivity or even inverted enantiopreference in the kinetic resolution of ethyl 3-phenylbutyrate.

Nasal mucus proteome and its involvement in allergic rhinitis.

Introduction: Nasal mucus is the first line defense barrier against various pathogens including allergens. Proteins in nasal mucus maybe used as biomarkers for diagnosis or future therapeutic strategies. Proteomics opens the possibility to investigate whole human proteomes.Areas Covered: We aimed to analyze the existing literature on nasal mucus and nasal secretions proteomic approaches especially in allergic rhinitis. A PubMed/Medline search was conducted entering the following keywords and combinations: "nasal mucus", "nasal lavage fluid," nasal secretions," "nasal swabs," "allergic rhinitis," "proteins," and "proteomics."Expert opinion: The majority of studies focus on single proteins or protein groups mainly using ELISA techniques. Four studies met the criteria using mass spectrometry in the analysis of nasal mucus proteomes in rhinologic diseases. In these studies, 7, 35, 267, and 430 proteins were identified, respectively. These four studies are discussed in this review and put in relation to seven other proteomic studies that focus on nasal lavage fluid and nasal secretions obtained by swabs or filter paper. To put it in a nutshell, proteomics facilitates the investigation of the nasal secretome and its role in healthy and diseased state and as potential biomarkers for new diagnostic or therapeutic approaches.

Endothelial lipase increases antioxidative capacity of high-density lipoprotein.

Endothelial lipase (EL) is a strong determinant of structural and functional properties of high-density lipoprotein (HDL). We examined whether the antioxidative capacity of HDL is affected by EL. EL-modified HDL (EL-HDL) and control EV-HDL were generated by incubation of HDL with EL- overexpressing or control HepG2 cells. As determined by native gradient gel electrophoresis, electron microscopy, and small-angle X-ray scattering EL-HDL is smaller than EV-HDL. Mass spectrometry revealed an enrichment of EL-HDL with lipolytic products and depletion of phospholipids and triacylglycerol. Kinetics of conjugated diene formation and HPLC-based malondialdehyde quantification revealed that EL-HDL exhibited a significantly higher resistance to copper ion-induced oxidation and a significantly higher capacity to protect low-density lipoprotein (LDL) from copper ion-induced oxidation when compared to EV-HDL. Depletion of the lipolytic products from EL-HDL abolished the capacity of EL-HDL to protect LDL from copper ion-induced oxidation, which could be partially restored by lysophosphatidylcholine enrichment. Proteomics of HDL incubated with oxidized LDL revealed significantly higher levels of methionine 136 sulfoxide in EL-HDL compared to EV-HDL. Chloramine T (oxidizes methionines and modifies free thiols), diminished the difference between EL-HDL and EV-HDL regarding the capacity to protect LDL from oxidation. In absence of LDL small EV-HDL and EL-HDL exhibited higher resistance to copper ion-induced oxidation when compared to respective large particles. In conclusion, the augmented antioxidative capacity of EL-HDL is primarily determined by the enrichment of HDL with EL-generated lipolytic products and to a lesser extent by the decreased HDL particle size and the increased activity of chloramine T-sensitive mechanisms.

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema.

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.

A Semi-Rationally Engineered Bacterial Pyrrolysyl-tRNA Synthetase Genetically Encodes Phenyl Azide Chemistry.

The site-specific incorporation of non-canonical amino acids (ncAAs) at amber codons requires an aminoacyl-tRNA synthetase and a cognate amber suppressor tRNA (tRNACUA ). The archaeal tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei have been extensively engineered to accept a versatile set of ncAAs. The PylRS/tRNACUA pair from the bacterium Desulfitobacterium hafniense is functional in Escherichia coli, however, variants of this PylRS have not been reported yet. In this study, the authors describe a bacterial PylRS from Desulfitobacterium hafniense, which the authors engineered for the reactive ncAA para-azido-l-phenylalanine (DhAzFRS) using a semi-rational approach. DhAzFRS preferred para-azido-l-phenylalanine to the canonical l-phenylalanine as the substrate. In addition, the authors demonstrate the functionality in E. coli of a hybrid DhAzFRS carrying the first 190 N-terminal amino acids of the Methanosarcina mazei PylRS. These results suggest that bacterial and archaeal PylRSs can be "mixed and matched" to tune their substrate specificity.

Characterization of a lipid droplet protein from Yarrowia lipolytica that is required for its oleaginous phenotype.

Oleaginous microorganisms are characterized by their ability to store high amounts of triacylglycerol (TAG) in intracellular lipid droplets (LDs). In this work, we characterized a protein of the oleaginous yeast Yarrowia lipolytica that is associated with LD and plays a role in the regulation of TAG storage. This protein is required for the oleaginous phenotype of Y. lipolytica because deletion of the coding gene results in a strongly reduced TAG content of the mutant. Therefore, we named it Oleaginicity Inducing LD protein, Oil1. Furthermore, a mutant overexpressing OIL1 accumulates more TAG than the wild type and is delayed in TAG lipolysis when this process is stimulated. We found that Oil1p plays a role in protecting the TAG content of the LD from degradation through lipases under conditions where the cell aims at building up its TAG reserves. Heterologous expression studies showed that Oil1p rescued the phenotype of a Saccharomyces cerevisiae mutant deleted for the perilipin-like protein Pln1p and that its expression in COS-7 cells resulted in increased TAG accumulation, similar to the phenotype of a perilipin 1 expressing control strain. Despite this phenotypical parallels to mammalian perilipins, Oil1p is not a member of this protein family and its activity does not depend on phosphorylation. Rather, our results suggest that ubiquitination might contribute to the function of Oil1p in Y. lipolytica and that a different mechanism evolved in this species to regulate TAG homeostasis.

Effect of Noncanonical Amino Acids on Protein-Carbohydrate Interactions: Structure, Dynamics, and Carbohydrate Affinity of a Lectin Engineered with Fluorinated Tryptophan Analogs.

Protein-carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated noncanonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan, which is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from Ralstonia solanacearum. We observed differences in stability and affinity toward fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate-aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors.

Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily.

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.