Structural basis for enzyme bifunctionality – the case of Gan1D from <i>Geobacillus stearothermophilus</i>

Lansky, Shifra; Zehavi, A.; Belrhali, Hassan; Shoham, Yuval; Shoham, G.

doi:10.1111/febs.14283

Cited by 10 publications

(15 citation statements)

References 98 publications

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“…A lactase é amplamente distribuída na natureza e pode ser isolada de fontes diferentes, tais como plantas (amêndoas, pêssegos, damascos, maçãs), órgãos de animais, leveduras, bactérias e fungos (RICHMOND; GRAY;STINE, 1981) As β-galactosidases de outras bactérias e leveduras têm tamanho de subunidade aproximadamente similar e podem ocorrer em forma dimérica (CHENG et al, 2012;RUTKIEWICZ-KROTEWICZ et al, 2016;LANSKY et al, 2017) ou tetramérica (PEREIRA-RODRÍGUEZ et al, 2012;WANG et al, 2015), em contraste com as β-galactosidases fúngicas, que são ativas como monômeros (MAKSIMAINEN et al, 2013;AGUSTÍN et al, 2017 DAAMEN, 2011). Contudo, diferentes espécies também vêm sendo estudadas tanto para hidrólise da lactose como para produção de galactooligossacarídeos, ou para ambos os processos (KAMRAN et al, 2016;CARDOSO et al, 2017;CHANALIA et al, 2018, respectivamente).…”

Section: Enzima β-Galactosidaseunclassified

Ciência e Tecnologia de Leite e Produtos Lácteos Sem Lactose

Dantas¹,

Verruck²,

Prudêncio³

2019

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Section: Enzima β-Galactosidaseunclassified

Ciência e Tecnologia de Leite e Produtos Lácteos Sem Lactose

Dantas¹,

Verruck²,

Prudêncio³

2019

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“…W420 forms a stacking interaction with the sugar ring. Figure 7B,C show the active site structures of two GH1 enzymes: 6-phospho-β-glucosidase/galactosidase Gan1D from Geobacillus stearothermophilus 30 (PDB code 4ZEN, sequence identity 44.6%), and β-glucosidase HoBGL from Halothermothrix orenii 28 , 29 (PDB code 4PTX, sequence identity 39.1%). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose

Higuchi

Matsufuji

Tanuma

et al. 2018

Sci Rep

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Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.

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“…We have attempted to link the loop with significant findings in literature. It is widely considered that a residue position in loop L8a, a conserved tryptophan in 6-P-β-galactosidases and alanine, phenylalanine, or methionine in 6-P-β-glucosidases (Ala423, Bl BglH numbering), is responsible for substrate specificity between the two 6-P-β-glycosidase activities. ,,, This residue position is in proximity to the O4 hydroxyl group of a bound substrate, the O4 hydroxyl group being the only difference between galacto- and gluco-derived substrates. Michalska and coauthors have gone on to determine that the axial arrangement of the O4 hydroxyl group in the galacto epimer would clash with the more closely situated Ala423 residue ( Bl BglH numbering) in the 6-P-β-glucosidase activity, and that this would prevent binding and catalysis.…”

Section: Resultsmentioning

confidence: 99%

“…This system is widespread in both Gram-positive and Gram-negative bacteria and catalyzes concomitant transport and phosphorylation of mono- and disaccharides, including cellobiose, lactose, sucrose, and trehalose. − In the cell, these phosphorylated disaccharides are mainly directed to glycolysis, and the first and determinant step to enter the pathway is the cleavage of the glycosidic bond promoted by glycoside hydrolases (GH), such as 6-phospho-beta-glucosidase (6-P-β-glucosidase) and 6-phospho-beta-galactosidase (6-P-β-galactosidase). Additionally, 6-P-β-glucosidases were already demonstrated to be involved in a cellobiose utilization system in Escherichia coli and other species, and 6-P-β-galactosidases were also shown to be part of lactose consumption systems in Lactobacillus casei and other lactic bacteria …”

Section: Introductionmentioning

confidence: 99%

X-ray Structure, Bioinformatics Analysis, and Substrate Specificity of a 6-Phospho-β-glucosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis

Veldman

Liberato

Almeida

et al. 2020

J. Chem. Inf. Model.

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In bacteria, mono-and disaccharides are phosphorylated during the uptake processes through the vastly spread transport system phosphoenolpyruvatedependent phosphotransferase. As an initial step in the phosphorylated disaccharide metabolism pathway, 6-phospho-β-glucosidases and 6-phospho-β-galactosidases play a crucial role by releasing phosphorylated and nonphosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. Here, an X-ray structure of a glycoside hydrolase family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglH, was determined at 2.2 Å resolution, and its substrate specificity was investigated. The sequence of BlBglH was compared to the sequences of 58 other GH1 enzymes using sequence alignments, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BlBglH was found to have sequence features characteristic of the 6-phospho-βglucosidase activity enzymes. Motif and structural observations highlighted the importance of loop L8 in 6-phospho-β-glucosidase activity enzymes. To further affirm enzyme specificity, molecular docking and molecular dynamics simulations were performed using the crystallographic structure of BlBglH. Docking was carried out with a 6-phospho-β-glucosidase enzyme activity positive and negative control ligand, followed by 400 ns of MD simulations. The positive and negative control ligands were PNP6Pglc and PNP6Pgal, respectively. PNP6Pglc maintained favorable interactions within the active site until the end of the MD simulation, while PNP6Pgal exhibited instability. The favorable binding of substrate stabilized the loops that surround the active site. Binding free energy calculations showed that the PNP6Pglc complex had a substantially lower binding energy compared to the PNP6Pgal complex. Altogether, the findings of this study suggest that BlBglH possesses 6-phospho-β-glucosidase enzymatic activity and revealed sequence and structural differences between bacterial GH1 enzymes of various activities.

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Structural basis for enzyme bifunctionality – the case of Gan1D from Geobacillus stearothermophilus

Cited by 10 publications

References 98 publications

Ciência e Tecnologia de Leite e Produtos Lácteos Sem Lactose

Ciência e Tecnologia de Leite e Produtos Lácteos Sem Lactose

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose

X-ray Structure, Bioinformatics Analysis, and Substrate Specificity of a 6-Phospho-β-glucosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis

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