Unprecedented Diversity of the Glycoside Hydrolase Family 70: A Comprehensive Analysis of Sequence, Structure, and Function.

The glycoside hydrolase family 70 (GH70) contains bacterial extracellular multidomain enzymes, synthesizing α-glucans from sucrose or starch-like substrates. A few dozen have been biochemically characterized, while crystal structures cover only the core domains and lack significant parts of auxiliary domains. Here we present a systematic overview of GH70 enzymes and their 3D structural organization and bacterial origin.

Polysaccharide utilization loci encoded DUF1735 likely functions as membrane-bound spacer for carbohydrate active enzymes.

Proteins featuring the Domain of Unknown Function 1735 are frequently found in Polysaccharide Utilization Loci, yet their role remains unknown. The domain and vicinity analyzer programs we developed mine the Kyoto Encyclopedia of Genes and Genomes and UniProt to enhance the functional prediction of DUF1735. Our datasets confirmed the exclusive presence of DUF1735 in Bacteroidota genomes, with Bacteroidetes thetaiotaomicron harboring 46 copies.

Tailor-Made α-Glucans by Engineering the Processivity of α-Glucanotransferases via Tunnel-Cleft Active Center Interconversions.

The function of polysaccharides is intimately associated with their size, which is largely determined by the processivity of transferases responsible for their synthesis. A tunnel active center architecture has been recognized as a key factor that governs processivity of several glycoside hydrolases (GHs), e.g.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg) from N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points.

Novel β-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154.

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo-β-galactosidase activity with regiopreference for hydrolyzing β-d-(1,6) glycosidic linkages.

Structural and time-resolved mechanistic investigations of protein hydrolysis by the acidic proline-specific endoprotease from Aspergillus niger.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role.

Crystal Structure of 4,6-α-Glucanotransferase GtfC-ΔC from Thermophilic 12AMOR1: Starch Transglycosylation in Non-Permuted GH70 Enzymes.

GtfC-type 4,6-α-glucanotransferase (α-GT) enzymes from Glycoside Hydrolase Family 70 (GH70) are of interest for the modification of starch into low-glycemic index food ingredients. Compared to the related GH70 GtfB-type α-GTs, found exclusively in lactic acid bacteria (LAB), GtfCs occur in non-LAB, share low sequence identity, lack circular permutation of the catalytic domain, and feature a single-segment auxiliary domain IV and auxiliary C-terminal domains. Despite these differences, the first crystal structure of a GtfC, GbGtfC-ΔC from 12AMOR1, and the first one representing a non-permuted GH70 enzyme, reveals high structural similarity in the core domains with most GtfBs, featuring a similar tunneled active site.

Characterization of the (Engineered) Branching Sucrase GtfZ-CD2 from Apilactobacillus kunkeei for Efficient Glucosylation of Benzenediol Compounds.

Branching sucrases, a subfamily of Glycoside Hydrolase family (GH70), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (α1→3) branching sucrase GtfZ-CD2 from DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e.

Crystal Structure of Levansucrase from the Gram-Negative Bacterium Provides Insights into Its Product Size Specificity.

Microbial levansucrases (LSs, EC 2.4.1.

Mutations in Amino Acid Residues of 121 GtfB 4,6-α-Glucanotransferase that Affect Reaction and Product Specificity.

121 4,6-α-glucanotransferase (Lr121 4,6-α-GTase), belonging to the glycosyl hydrolase (GH) 70 GtfB subfamily, converts starch and maltodextrins into linear isomalto/malto polysaccharides (IMMPs) with consecutive (α1 → 6) linkages. The recent elucidation of its crystal structure allowed identification and analysis of further structural features that determine its reaction and product specificity. Herein, sequence alignments between GtfB enzymes with different product linkage specificities (4,6-α-GTase and 4,3-α-GTase) identified amino acid residues in GH70 homology motifs, which may be critical for reaction and product specificity.

Enzymatic glucosylation of polyphenols using glucansucrases and branching sucrases of glycoside hydrolase family 70.

Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols.

Insights into Broad-Specificity Starch Modification from the Crystal Structure of NCC 2613 4,6-α-Glucanotransferase GtfB.

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase ( NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB ( NCC 2970) and show that they are able to convert both linear and branched starch substrates.

Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T, a halophilic archaeal strain.

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form and with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose).

Identification of Thermotoga maritima MSB8 GH57 α-amylase AmyC as a glycogen-branching enzyme with high hydrolytic activity.

AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary structural comparison suggest that AmyC could actually be a glycogen-branching enzyme (GBE), a key enzyme in the biosynthesis of glycogen. This communication presents phylogenetic and biochemical evidence that AmyC is a GBE with a relatively high hydrolytic (α-amylase) activity (up to 30% of the total activity), creating a branched α-glucan with 8.

Enantioselective Synthesis of Pharmaceutically Active γ-Aminobutyric Acids Using a Tailor-Made Artificial Michaelase in One-Pot Cascade Reactions.

Chiral γ-aminobutyric acid (GABA) analogues represent abundantly prescribed drugs, which are broadly applied as anticonvulsants, as antidepressants, and for the treatment of neuropathic pain. Here we report a one-pot two-step biocatalytic cascade route for synthesis of the pharmaceutically relevant enantiomers of γ-nitrobutyric acids, starting from simple precursors (acetaldehyde and nitroalkenes), using a tailor-made highly enantioselective artificial "Michaelase" (4-oxalocrotonate tautomerase mutant L8Y/M45Y/F50A), an aldehyde dehydrogenase with a broad non-natural substrate scope, and a cofactor recycling system. We also report a three-step chemoenzymatic cascade route for the efficient chemical reduction of enzymatically prepared γ-nitrobutyric acids into GABA analogues in one pot, achieving high enantiopurity (e.

Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate.

Glucansucrase Gtf180-ΔN from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (α1→2)/(α1→3)/(α1→4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (α1→3) linkage and increased F4 4',2-glc-lac with (α1→4)/(α1→2) linkages.

Biochemical characterization of a GH70 protein from Lactobacillus kunkeei DSM 12361 with two catalytic domains involving branching sucrase activity.

The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ).

Biochemical and genetic characterization of fungal proline hydroxylase in echinocandin biosynthesis.

An intriguing structural feature of echinocandins is the incorporation of hydroxylated amino acids. Elucidation of the machinery and the mechanism responsible for this modification is critical to generate new echinocandin derivatives with enhanced antifungal activity. In our present study, we biochemically characterized the α-ketoglutarate/Fe-dependent proline hydroxylase (HtyE) from two Aspergillus species, Aspergillus pachycristatus and Aspergillus aculeatus, in the respective echinocandin B and aculeacin A biosynthetic gene clusters.

Biochemical characterization of two GH70 family 4,6-α-glucanotransferases with distinct product specificity from Lactobacillus aviarius subsp. aviarius DSM 20655.

Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviarius subsp. aviarius DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV.

Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose.

Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical properties are produced, which have found diverse (potential) commercial applications, e.g.

A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy.

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPF.

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.

Biochemical Characterization of the Functional Roles of Residues in the Active Site of the β-Galactosidase from Bacillus circulans ATCC 31382.

The β-galactosidase enzyme from Bacillus circulans ATCC 31382 BgaD is widely used in the food industry to produce prebiotic galactooligosaccharides (GOS). Recently, the crystal structure of a C-terminally truncated version of the enzyme (BgaD-D) has been elucidated. The roles of active site amino acid residues in β-galactosidase enzyme reaction and product specificity have remained unknown.

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad.

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia.