The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases

One of the most intriguing groups of enzymes, the feruloyl esterases (FAEs), is ubiquitous in both simple and complex organisms. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing high-added value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production and partial characterization of FAEs from fungi, while much less is known about FAEs of bacterial or plant origin. Initial classification studies on FAEs were restricted on sequence similarity and substrate specificity on just four model substrates and considered only a handful of FAEs belonging to the fungal kingdom. This study centers on the descriptor-based classification and structural analysis of experimentally verified and putative FAEs; nevertheless, the framework presented here is applicable to every poorly characterized enzyme family. 365 FAE-related sequences of fungal, bacterial and plantae origin were collected and they were clustered using Self Organizing Maps followed by k-means clustering into distinct groups based on amino acid composition and physico-chemical composition descriptors derived from the respective amino acid sequence. A Support Vector Machine model was subsequently constructed for the classification of new FAEs into the pre-assigned clusters. The model successfully recognized 98.2% of the training sequences and all the sequences of the blind test. The underlying functionality of the 12 proposed FAE families was validated against a combination of prediction tools and published experimental data. Another important aspect of the present work involves the development of pharmacophore models for the new FAE families, for which sufficient information on known substrates existed. Knowing the pharmacophoric features of a small molecule that are essential for binding to the members of a certain family opens a window of opportunities for tailored applications of FAEs

The gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD. METHODS: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D Lombard et al. (2014) H/ Machius et al. (1995) C NMR spectroscopy and enzymatic degradation studies. RESULTS: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose. CONCLUSIONS: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes. GENERAL SIGNIFICANCE: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient

Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract

The glycemic carbohydrates we consume are currently viewed in an unfavorable light in both the consumer and medical research worlds. In significant part, these carbohydrates, mainly starch and sucrose, are looked upon negatively due to their rapid and abrupt glucose delivery to the body which causes a high glycemic response. However, dietary carbohydrates which are digested and release glucose in a slow manner are recognized as providing health benefits. Slow digestion of glycemic carbohydrates can be caused by several factors, including food matrix effect which impedes α-amylase access to substrate, or partial inhibition by plant secondary metabolites such as phenolic compounds. Differences in digestion rate of these carbohydrates may also be due to their specific structures (e.g. variations in degree of branching and/or glycosidic linkages present). In recent years, much has been learned about the synthesis and digestion kinetics of novel α-glucans (i.e. small oligosaccharides or larger polysaccharides based on glucose units linked in different positions by α-bonds). It is the synthesis and digestion of such structures that is the subject of this review

Discovery and development of novel glucanotransferases for healthier foods

Providing nutritious, healthy and sustainably produced food is one of the main objectives of food companies such as Nestlé. Slowly digestible carbohydrates and dietary fibers are considered beneficial for human health. The aim of this work was to expand the enzymatic toolbox and to develop enzyme processes that reduce the glycaemic index of starch-containing foods. By mining the gene pool of the Nestlé culture collection (NCC) with more than 3000 food grade strains, we identified sequences for novel glucanotransferases (Gtf). Two enzymes belonging to the sub family GtfB of the glycosyl hydrolase family 70 (GH70) originating from Lactobacillus reuteri (NCC 2613) and L. fermentum (NCC 2970) as well as the GtfD enzymes from Paenibacillus beijingenis and Azobacter chroococcum (of non-NCC origin) were expressed and biochemically characterized. All four Gtf enzymes produce unique α-glucans with alternating α(1,4) and α(1,6) or α(1,4) and α(1,3) linkages of different molecular size. In vitro digestion and process development studies were performed using raw materials (e.g. wheat flour) to evaluate the impact on starch digestibility as well as the in-process modification of cereal products

Structural Characterisation by ESI-MS of Feruloylated Arabino-oligosaccharides Synthesised by Chemoenzymatic Esterification

The chemoenzymatic synthesis of feruloylated arabino-oligosaccharides has neen achieved, using a feruloyl esterase type C from Sporotrichum themophile (StFaeC). The structure of the feruloyl products was comfirmed by ESI-MS

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures

Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database:The Lactobacillus reuteri NCC 2613 GtfB

The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving (α1 → 4)-linkages in maltodextrins/starch and synthesizing new (α1 → 6)-linkages. In this work, 106 putative GtfBs were identified in the Nestlé Culture Collection genome database with ~2700 genomes, and the L. reuteri NCC 2613 one was selected for further characterization based on variations in its conserved motifs. Using amylose the L. reuteri NCC 2613 GtfB synthesizes a low-molecular-mass reuteran-like polymer consisting of linear (α1 → 4) sequences interspersed with (α1 → 6) linkages, and (α1 → 4,6) branching points. This product specificity is novel within the GtfB subfamily, mostly comprising 4,6-α-glucanotransferases synthesizing consecutive (α1 → 6)-linkages. Instead, its activity resembles that of the GtfD 4,6-α-glucanotransferases identified in non-LAB strains. This study demonstrates the potential of large-scale genome sequence data for the discovery of enzymes of interest for the food industry. The L. reuteri NCC 2613 GtfB is a valuable addition to the starch-converting GH70 enzyme toolbox. It represents a new evolutionary intermediate between families GH13 and GH70, and provides further insights into the structure-function relationships of the GtfB subfamily enzymes

Combining Substrate Specificity Analysis with Support Vector Classifiers Reveals Feruloyl Esterase as a Phylogenetically Informative Protein Group

Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able to hydrolyze the ester bonds linking ferulic acid to plant cell wall polysaccharides. The diversity of substrate specificities found in the FAE family shows that this family is old enough to have experienced the emergence and loss of many activities. In this study we evaluate the relative activity of FAEs against a variety of model substrates as a novel predictive tool for Ascomycota taxonomic classification. Our approach consists of two analytical steps; (1) an initial unsupervised analysis to cluster the FAEs substrate specificity data which were generated by cultivation of 34 Ascomycota strains and then an analysis of the produced enzyme cocktail against 10 substituted cinnamate and phenylalkanoate methyl esters, (2) a second, supervised analysis for training a predictor built on these substrate activities. By applying both linear and non-linear models we were able to correctly predict the taxonomic Class (∼86% correct classification), Order (∼88% correct classification) and Family (∼88% correct classification) that the 34 Ascomycota belong to, using the activity profiles of the FAEs. The good correlation with the FAEs substrate specificities that we have defined via our phylogenetic analysis not only suggests that FAEs are phylogenetically informative proteins but it is also a considerable step towards improved FAEs functional prediction.published_or_final_versio

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes, inactive on sucrose but using maltodextrins/starch as substrates, have been established (e.g. GtfB of Lactobacillus reuteri 121). Compared to the broad linkage specificity found in GSs, all GH70 starch-acting enzymes characterized so far possess 4,6-α-glucanotransferase activity, cleaving (α1 → 4)-linkages and synthesizing new (α1 → 6)-linkages. In this work a gene encoding a putative GH70 family enzyme was identified in the genome of Lactobacillus fermentum NCC 2970, displaying high sequence identity with L. reuteri 121 GtfB 4,6-α-glucanotransferase, but also with unique variations in some substrate-binding residues of GSs. Characterization of this L. fermentum GtfB and its products revealed that it acts as a 4,3-α-glucanotransferase, converting amylose into a new type of α-glucan with alternating (α1 → 3)/(α 1 → 4)-linkages and with (α1 → 3,4) branching points. The discovery of this novel reaction specificity in GH70 family and clan GH-H expands the range of α-glucans that can be synthesized and allows the identification of key positions governing the linkage specificity within the active site of the GtfB-like GH70 subfamily of enzymes