scholarly journals Amylolytic enzymes - focus on the alpha-amylases from Archae and plants

2021 ◽  
Vol 9 (1) ◽  
pp. 5-26
Author(s):  
Štefan Janeček
Keyword(s):  
Three Dimensional ◽  
Structural Features ◽  
Catalytic Triad ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolases ◽  
Amylolytic Enzymes ◽  
Conserved Sequence ◽  
Glycosidic Bonds ◽  
Evolutionary Relatedness ◽  
Tim Barrel

Amylolytic enzymes represent a group of starch hydrolases and related enzymes that are active towards the α-glycosidic bonds in starch and related poly- and oligosaccharides. The three best known amylolytic enzymes are α-amylase, β-amylase and glucoamylase that, however, differ from each other by their amino acid sequences, three-dimensional structures, reaction mechanisms and catalytic machineries. In the sequence-based classification of all glycoside hydrolases (GHs) they have therefore been classified into the three independent families: GH13 (α-amylases), GH14 (β-amylases) and GH15 (glucoamylases). Some amylolytic enzymes have been placed to the families GH31 and GH57. The family GH13 together with the families GH70 and GH77 constitutes the clan GH-H, well-known as the α-amylase family. It contains more than 6,000 sequences and covers 30 various enzyme specificities sharing the conserved sequence regions, catalytic TIM-barrel fold, retaining reaction mechanism and catalytic triad. Among the GH13 α-amylases, those produced by plants and archaebacteria exhibit common sequence similarities that distinguish them from the α-amylases of the remaining taxonomic sources. Despite the close evolutionary relatedness between the plant and archaeal α-amylases, there are also specific differences that discriminate them from each other. These specific differences could be used in an effort to reveal the sequence-structural features responsible for the high thermostability of the α-amylases from Archaea.

A novel GH13 subfamily of α-amylases with a pair of tryptophans in the helix α3 of the catalytic TIM-barrel, the LPDlx signature in the conserved sequence region V and a conserved aromatic motif at the C-terminus

Biologia ◽  
2015 ◽  
Vol 70 (10) ◽  
Author(s):  
Štefan Janeček ◽  
Andrea Kuchtová ◽  
Soňa Petrovičová
Keyword(s):  
Structural Features ◽  
Point Of View ◽  
Enzyme Specificity ◽  
Entire Family ◽  
Amylolytic Enzymes ◽  
Conserved Sequence ◽  
C Terminus ◽  
Cazy Database ◽  
Tim Barrel ◽  
Evolutionary Comparison

AbstractThe α-amylase enzyme specificity has been classified in the Carbohydrate-Active enZyme (CAZy) database into the families GH13, GH57, GH119 and eventually also GH126. α-Amylase is a glycoside hydrolase (GH) that catalyses in an endo-fashion the hydrolysis of the α-1,4-glucosidic linkages in starch and related α-glucans employing the retaining reaction mechanism. The family GH13 is the main α-amylase family with more than 28,000 members and 30 different specificities. The entire family GH13 has already been divided into 40 subfamilies; the α-amylase enzyme specificity being found in the subfamilies GH13 1, 5, 6, 7, 15, 19, 24, 27, 28, 32, 36 and 37. The present in silico study delivers a proposal to create a novel GH13 subfamily with the specificity of α-amylase. The proposal is based on a detailed bioinformatics analysis consisting of sequence, structural and evolutionary comparison of experimentally characterized α-amylases from, e.g., Bacillus aquimaris, Anoxybacillus sp. SK3-4 and DT3-1 and Geobacillus thermoleovorans, and hypothetical proteins, accompanied by α-amylases from well-established GH13 subfamilies and by closely related amylolytic enzymes (mainly from the subfamily GH13 31). Three sequence-structural features can be ascribed to the members of the newly proposed GH13 subfamily: (i) the pair of adjacent tryptophan residues positioned between the CSR-V and CSR-II in the helix α3 of the catalytic TIM-barrel; (ii) the sequence LPDlx in their CSR-V; and (iii) a ~30-residue long C-terminal region with a motif of five conserved aromatic residues. From the evolutionary point of view, the novel GH13 α-amylase subfamily is most closely related to fungal and yeast α-amylases classified in the subfamily GH13_1.

scholarly journals In Silico Analysis of Fungal and Chloride-Dependent α-Amylases within the Family GH13 with Identification of Possible Secondary Surface-Binding Sites

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5704
Author(s):  
Zuzana Janíčková ◽  
Štefan Janeček
Keyword(s):  
Binding Sites ◽  
In Silico ◽  
Homo Sapiens ◽  
In Silico Analysis ◽  
Surface Binding ◽  
Conserved Sequence ◽  
Evolutionary Relatedness ◽  
The Family ◽  
Tim Barrel ◽  
Silico Analysis

This study brings a detailed bioinformatics analysis of fungal and chloride-dependent α-amylases from the family GH13. Overall, 268 α-amylase sequences were retrieved from subfamilies GH13_1 (39 sequences), GH13_5 (35 sequences), GH13_15 (28 sequences), GH13_24 (23 sequences), GH13_32 (140 sequences) and GH13_42 (3 sequences). Eight conserved sequence regions (CSRs) characteristic for the family GH13 were identified in all sequences and respective sequence logos were analysed in an effort to identify unique sequence features of each subfamily. The main emphasis was given on the subfamily GH13_32 since it contains both fungal α-amylases and their bacterial chloride-activated counterparts. In addition to in silico analysis focused on eventual ability to bind the chloride anion, the property typical mainly for animal α-amylases from subfamilies GH13_15 and GH13_24, attention has been paid also to the potential presence of the so-called secondary surface-binding sites (SBSs) identified in complexed crystal structures of some particular α-amylases from the studied subfamilies. As template enzymes with already experimentally determined SBSs, the α-amylases from Aspergillus niger (GH13_1), Bacillus halmapalus, Bacillus paralicheniformis and Halothermothrix orenii (all from GH13_5) and Homo sapiens (saliva; GH13_24) were used. Evolutionary relationships between GH13 fungal and chloride-dependent α-amylases were demonstrated by two evolutionary trees—one based on the alignment of the segment of sequences spanning almost the entire catalytic TIM-barrel domain and the other one based on the alignment of eight extracted CSRs. Although both trees demonstrated similar results in terms of a closer evolutionary relatedness of subfamilies GH13_1 with GH13_42 including in a wider sense also the subfamily GH13_5 as well as for subfamilies GH13_32, GH13_15 and GH13_24, some subtle differences in clustering of particular α-amylases may nevertheless be observed.

scholarly journals Identification of fibrillogenic regions in human triosephosphate isomerase

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1676 ◽  
Author(s):  
Edson N. Carcamo-Noriega ◽  
Gloria Saab-Rincon
Keyword(s):  
Triosephosphate Isomerase ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Structural Features ◽  
Structural Topology ◽  
Tim Barrel ◽  
Functional Amyloids ◽  
Polypeptide Chains ◽  
Protein Fragmentation ◽  
Chain Interaction

Background.Amyloid secondary structure relies on the intermolecular assembly of polypeptide chains through main-chain interaction. According to this, all proteins have the potential to form amyloid structure, nevertheless, in nature only few proteins aggregate into toxic or functional amyloids. Structural characteristics differ greatly among amyloid proteins reported, so it has been difficult to link the fibrillogenic propensity with structural topology. However, there are ubiquitous topologies not represented in the amyloidome that could be considered as amyloid-resistant attributable to structural features, such is the case of TIM barrel topology.Methods.This work was aimed to study the fibrillogenic propensity of human triosephosphate isomerase (HsTPI) as a model of TIM barrels. In order to do so, aggregation of HsTPI was evaluated under native-like and destabilizing conditions. Fibrillogenic regions were identified by bioinformatics approaches, protein fragmentation and peptide aggregation.Results.We identified four fibrillogenic regions in the HsTPI corresponding to theβ3,β6,β7y α8 of the TIM barrel. From these, theβ3-strand region (residues 59–66) was highly fibrillogenic. In aggregation assays, HsTPI under native-like conditions led to amorphous assemblies while under partially denaturing conditions (urea 3.2 M) formed more structured aggregates. This slightly structured aggregates exhibited residual cross-βstructure, as demonstrated by the recognition of the WO1 antibody and ATR-FTIR analysis.Discussion.Despite the fibrillogenic regions present in HsTPI, the enzyme maintained under native-favoring conditions displayed low fibrillogenic propensity. This amyloid-resistance can be attributed to the three-dimensional arrangement of the protein, whereβ-strands, susceptible to aggregation, are protected in the core of the molecule. Destabilization of the protein structure may expose inner regions promotingβ-aggregation, as well as the formation of hydrophobic disordered aggregates. Being this last pathway kinetically favored over the thermodynamically more stable fibril aggregation pathway.

scholarly journals A Novel Mercuric Reductase from the Unique Deep Brine Environment of Atlantis II in the Red Sea

2013 ◽  
Vol 289 (3) ◽  
pp. 1675-1687 ◽  
Author(s):  
Ahmed Sayed ◽  
Mohamed A. Ghazy ◽  
Ari J. S. Ferreira ◽  
João C. Setubal ◽  
Felipe S. Chambergo ◽  
...  
Keyword(s):  
Red Sea ◽  
Three Dimensional ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Mercuric Reductase ◽  
Three Dimensional Modeling ◽  
High Concentrations ◽  
Convective Layer

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg2+, and efficiently detoxifies Hg2+in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

scholarly journals Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications

Amylase ◽  
2018 ◽  
Vol 2 (1) ◽  
pp. 44-57
Author(s):  
Tulasi Satyanarayana ◽  
Mohanan Nisha
Keyword(s):  
Catalytic Domain ◽  
Bread Making ◽  
Amylolytic Enzymes ◽  
Conserved Sequence ◽  
Glycosidic Bonds ◽  
Bifunctional Enzymes ◽  
One Step ◽  
Starch Saccharification ◽  
Characteristic Features ◽  
The Cost

AbstractAmylopullulanases are endoacting bifunctional enzymes capable of hydrolyzing α-1,4- and α-1,6-glycosidic linkages in starch, amylose, pullulan, amylopectin and related oligosaccharides. These enzymes possess single or dual active site(s) for cleaving α-1,4- and α-1,6-glycosidic bonds; the former are called amylopullulanases, and the latter, α-amylase-pullulanases. These are grouped into GH13 and GH57 families based on the architecture of the catalytic domain and the number of conserved sequence regions. The amylopullulanases/α-amylasepullulanases are produced by bacteria as well as archaea, and among them, thermophilic and hyperthermophilic species are the major producers. The thermostable amylopullulanases find application in one-step starch liquefaction-saccharification to form various sugar syrups and maltooligosaccharides. The starch saccharification process catalysed by amylopullulanases minimizes the use of other amylolytic enzymes, like α-amylase and glucoamylase, thereby reducing the cost of sugar syrups. The enzymes also find applications in bread making as an anti-stale and as a detergent additive.

scholarly journals Improving Subcellular Protein Location Classification by Incorporating Three-Dimensional Structure Information

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1607
Author(s):  
Ge Wang ◽  
Yu-Jia Zhai ◽  
Zhen-Zhen Xue ◽  
Ying-Ying Xu
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Machine Learning Algorithms ◽  
Dimensional Structure ◽  
Data Types ◽  
Functional Protein ◽  
Structure Information ◽  
Protein Location

The subcellular locations of proteins are closely related to their functions. In the past few decades, the application of machine learning algorithms to predict subcellular protein locations has been an important topic in proteomics. However, most studies in this field used only amino acid sequences as the data source. Only a few works focused on other protein data types. For example, three-dimensional structures, which contain far more functional protein information than sequences, remain to be explored. In this work, we extracted various handcrafted features to describe the protein structures from physical, chemical, and topological aspects, as well as the learned features obtained by deep neural networks. We then used these features to classify the subcellular protein locations. Our experimental results demonstrated that some of these structural features have a certain effect on the protein location classification, and can help improve the performance of sequence-based location predictors. Our method provides a new view for the analysis of protein spatial distribution, and is anticipated to be used in revealing the relationships between protein structures and functions.

scholarly journals A conserved folding nucleus sculpts the free energy landscape of bacterial and archaeal orthologs from a divergent TIM barrel family

2021 ◽  
Vol 118 (17) ◽  
pp. e2019571118
Author(s):  
Rohit Jain ◽  
Khaja Muneeruddin ◽  
Jeremy Anderson ◽  
Michael J. Harms ◽  
Scott A. Shaffer ◽  
...  
Keyword(s):  
Free Energy ◽  
Dynamic Equilibrium ◽  
High Energy ◽  
Amino Acid Sequences ◽  
Intact Protein ◽  
Free Energy Surface ◽  
Conserved Sequence ◽  
Evolutionary Forces ◽  
Tim Barrel ◽  
Hdx Ms

The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (βα)1–8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a ∼3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen–deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (α1β2) and a large cluster (β5α5β6α6β7) and that these clusters form cores of stability in Ia and Ibp. The strongest protection in both states resides in β4α4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising α-helix propensity for β4, preceded by a highly conserved βα-hairpin clamp that links β3 and β4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins.

scholarly journals Extension of the taxonomic coverage of the family GH126 outside Firmicutes and in silico characterization of its non-catalytic terminal domains

3 Biotech ◽  
2020 ◽  
Vol 10 (10) ◽  
Author(s):  
Lenka Kerényiová ◽  
Štefan Janeček
Keyword(s):  
In Silico ◽  
Homology Modelling ◽  
Family Members ◽  
Three Dimensional ◽  
In Silico Analysis ◽  
Glycoside Hydrolases ◽  
Modular Organization ◽  
Conserved Sequence ◽  
The Family

Abstract The family GH126 is a family of glycoside hydrolases established in 2011. Officially, in the CAZy database, it counts ~ 1000 sequences originating solely from bacterial phylum Firmicutes. Two members, the proteins CPF_2247 from Clostridium perfringens and PssZ from Listeria monocytogenes have been characterized as a probable α-amylase and an exopolysaccharide-specific glycosidase, respectively; their three-dimensional structures being also solved as possessing catalytic (α/α)6-barrel fold. Previously, based on a detailed in silico analysis, the seven conserved sequence regions (CSRs) were identified for the family along with elucidating basic evolutionary relationships within the family members. The present study represents a continuation study focusing on two particular aims: (1) to find out whether the taxonomic coverage of the family GH126 might be extended outside the Firmicutes and, if positive, to deliver those out-of-Firmicutes proteins with putting them into the context of the family; and (2) to identify the family members containing the N- and/or C-terminal extensions of their polypeptide chain, additional to the catalytic (α/α)6-barrel domain, and perform the bioinformatics characterization of the extra domains. The main results could be summarized as follows: (1) 17 bacterial proteins caught by BLAST searches outside Firmicutes (especially from phyla Proteobacteria, Actinobacteria and Bacteroidetes) have been found and convincingly suggested as new family GH126 members; and (2) a thioredoxin-like fold and various leucine-rich repeat motifs identified by Phyre2 structure homology modelling have been recognized as extra domains occurring most frequently in the N-terminal extensions of family GH126 members possessing a modular organization.

scholarly journals A pair of esterases from a commensal gut bacterium remove acetylations from all positions on complex β-mannans

2020 ◽  
Vol 117 (13) ◽  
pp. 7122-7130 ◽  
Author(s):  
Leszek Michalak ◽  
Sabina Leanti La Rosa ◽  
Shaun Leivers ◽  
Lars Jordhøy Lindstad ◽  
Åsmund Kjendseth Røhr ◽  
...  
Keyword(s):  
High Performance ◽  
Plant Cell Wall ◽  
3D Structure ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Glycoside Hydrolases ◽  
Time Resolved ◽  
Mannose Residue ◽  
Insight Into

β-mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. β-mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs); however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, RiCE2 and RiCE17, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of RiCE17 with a mannopentaose in the active site shows that the CBM35 domain of RiCE17 forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points toward the Ser41 of the catalytic triad. Cavities on the RiCE17 surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite −1 and +1). In-depth characterization of the two enzymes using time-resolved NMR, high-performance liquid chromatography (HPLC), and mass spectrometry demonstrates that they work in a complementary manner. RiCE17 exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O-, and 6-O-acetylations. Activity of RiCE2 is dependent on RiCE17 removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O-specific RiCE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides.

scholarly journals Cyclodextrin-preferring glycoside hydrolases: properties and applications

Amylase ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 23-37
Author(s):  
Iqra Aroob ◽  
Nasir Ahmad ◽  
Naeem Rashid
Keyword(s):  
Substrate Specificity ◽  
Three Dimensional ◽  
Distinct Group ◽  
Structural Features ◽  
Biochemical Properties ◽  
Glycoside Hydrolases ◽  
Cazy Database ◽  
Potential Applications ◽  
Pharmaceutical Industries ◽  
Hydrolyzing Enzymes

Abstract Cyclodextrin-hydrolyzing enzymes are widespread in bacteria and archaea where they play their roles in carbohydrates metabolism. They were previously characterized as cyclodextrinases, neopullulanases and maltogenic amylases. In the Carbohydrate-Active enZyme (CAZy) database, most of these enzymes are grouped into the GH13_20 subfamily of the α-amylase family GH13. Here, we have summarized the information available on the substrate specificity, structural features, physiological roles and applications of cyclodextrin-preferring glycoside hydrolases. These enzymes form a distinct group in the α-amylase family. Members of this distinct group possess an extra extension at the N-terminus, which causes a modification of the active site geometry thus making these enzymes more specific for smaller molecules like cyclodextrins than for macromolecules such as starches or pullulan. Multi-substrate specificity, hydrolytic as well as transglycosylation activities make these enzymes attractive for applications in the food and pharmaceutical industries. We have tried here to collect information available on their biochemical properties, three-dimensional structures, physiological roles and potential applications.

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