scholarly journals Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6586
Author(s):  
Rodrigo A. Arreola-Barroso ◽  
Alexey Llopiz ◽  
Leticia Olvera ◽  
Gloria Saab-Rincón
Keyword(s):  
Glycoside Hydrolase ◽  
Thermotoga Maritima ◽  
Computational Approach ◽  
Glycoside Hydrolase Family ◽  
Structural Determinants ◽  
Dynamic Simulations ◽  
Reaction Specificity ◽  
Essential Contribution ◽  
Hydrolysis Of ◽  
Hydrolase Family

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

scholarly journals Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yuya Kuritani ◽  
Kohei Sato ◽  
Hideo Dohra ◽  
Seiichiro Umemura ◽  
Motomitsu Kitaoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metabolic Pathway ◽  
Glycoside Hydrolase ◽  
Gene Locus ◽  
Glycoside Hydrolase Family ◽  
Sequential Reaction ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Hydrolase Family

AbstractLevoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-d-glucose by LgdB2; next, LgdC reduces 3-keto-d-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

Bacillus licheniformistrehalose-6-phosphate hydrolase structures suggest keys to substrate specificity

2016 ◽  
Vol 72 (1) ◽  
pp. 59-70 ◽  
Author(s):  
Min-Guan Lin ◽  
Meng-Chun Chi ◽  
Vankadari Naveen ◽  
Yi-Ching Li ◽  
Long-Liu Lin ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Bacillus Licheniformis ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Positive Charge ◽  
Glycoside Hydrolase Family ◽  
Erwinia Rhapontici ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures ofBacillus licheniformisTreA (BlTreA) and its R201Q mutant complexed withp-nitrophenyl-α-D-glucopyranoside (R201Q–pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure ofBlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site ofBlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions ofBacillus cereusoligo-1,6-glucosidase (BcOgl) andErwinia rhaponticiisomaltulose synthase (NX-5), the surface potential of theBlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity ofBlTreA. Strikingly, the281HHLK284motif and Lys292 play critical roles in substrate discrimination byBlTreA.

scholarly journals Two Novel α-L-Arabinofuranosidases from Bifidobacterium longum subsp. longum Belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Masahiro Komeno ◽  
Honoka Hayamizu ◽  
Kiyotaka Fujita ◽  
Hisashi Ashida
Keyword(s):  
Gene Cluster ◽  
Glycoside Hydrolase ◽  
Bifidobacterium Longum ◽  
Glycoside Hydrolase Family ◽  
Divalent Metal Ions ◽  
Content Type ◽  
Glycoside Hydrolase Family 43 ◽  
Genes Encoding ◽  
Hydrolysis Of ◽  
Hydrolase Family

ABSTRACT Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited α-l-arabinofuranosidase activity toward p-nitrophenyl-α-l-arabinofuranoside but no activity toward p-nitrophenyl-β-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of α1,2- and α1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the α1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan. IMPORTANCE We identified two novel α-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on α1,2/3-l-arabinofuranosyl linkages, while the latter acts on the α1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. The genes encoding these enzymes are located side by side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.

scholarly journals Structural studies of a glycoside hydrolase family 3 β-glucosidase from the model fungusNeurospora crassa

2018 ◽  
Vol 74 (12) ◽  
pp. 787-796 ◽  
Author(s):  
Saeid Karkehabadi ◽  
Henrik Hansson ◽  
Nils Egil Mikkelsen ◽  
Steve Kim ◽  
Thijs Kaper ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Crystalline Cellulose ◽  
Glycoside Hydrolase Family ◽  
Special Importance ◽  
Biologically Relevant ◽  
Sequence Identity ◽  
Active Site Cleft ◽  
Dimeric Form ◽  
Hydrolysis Of ◽  
Hydrolase Family

The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A fromNeurospora crassa(NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form ofNcCel3A is reported. The structure has been refined to 2.25 Å resolution, with anRcrystandRfreeof 0.18 and 0.22, respectively.NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity withHypocrea jecorinaCel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β-glucosidase fromRasamsonia emersonii.NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 β-glucosidases and account for the high cellobiose specificity of this subfamily.

scholarly journals A computational approach to structural properties of glycoside hydrolase family 4 from bacteria.

2013 ◽  
Vol 60 (4) ◽  
Author(s):  
Dana Craciun ◽  
Beatrice Vlad-Oros ◽  
Nicoleta Filimon ◽  
Vasile Ostafe ◽  
Adriana Isvoran
Keyword(s):  
Protein Interactions ◽  
Thermotoga Maritima ◽  
Structural Characteristics ◽  
Specific Protein ◽  
Computational Approach ◽  
Glycoside Hydrolase Family ◽  
Protein Protein Interactions ◽  
Sequence Identity ◽  
Enzyme Family ◽  
Hydrolase Family

Structural bioinformatics approaches applied to the alpha- and beta-glycosidases from the GH4 enzyme family reveal that, despite low sequence identity, these enzymes possess quite similar global structural characteristics reflecting a common reaction mechanism. Locally, there are a few distinctive structural characteristics of GH4 alpha- and beta-glycosidases, namely, surface cavities with different geometric characteristics and two regions with highly dissimilar structural organizations and distinct physicochemical properties in the alpha- and beta-glucosidases from Thermotoga maritima. We suggest that these structurally dissimilar regions may be involved in specific protein-protein interactions and this hypothesis is sustained by the predicted distinct functional partners of the investigated proteins. Also, we predict that alpha- and beta-glycosidases from the GH4 enzyme family interact with difenoconazole, a fungicide, but there are different features of these interactions especially concerning the identified structurally distinct regions of the investigated proteins.

scholarly journals CalkGH9T: A Glycoside Hydrolase Family 9 Enzyme from Clostridium alkalicellulosi

Catalysts ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1011
Author(s):  
Paripok Phitsuwan ◽  
Sengthong Lee ◽  
Techly San ◽  
Khanok Ratanakhanokchai
Keyword(s):  
Glycoside Hydrolase ◽  
Cellulose Degradation ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Type I ◽  
Amorphous Cellulose ◽  
Carbohydrate Binding Modules ◽  
Glycoside Hydrolase Family 9 ◽  
Hydrolysis Of ◽  
Hydrolase Family

Glycoside hydrolase family 9 (GH9) endoglucanases are important enzymes for cellulose degradation. However, their activity on cellulose is diverse. Here, we cloned and expressed one GH9 enzyme (CalkGH9T) from Clostridium alkalicellulosi in Escherichia coli. CalkGH9T has a modular structure, containing one GH9 catalytic module, two family 3 carbohydrate binding modules, and one type I dockerin domain. CalkGH9T exhibited maximal activity at pH 7.0–8.0 and 55 °C and was resistant to urea and NaCl. It efficiently hydrolyzed carboxymethyl cellulose (CMC) but poorly degraded regenerated amorphous cellulose (RAC). Despite strongly binding to Avicel, CalkGH9T lacked the ability to hydrolyze this substrate. The hydrolysis of CMC by CalkGH9T produced a series of cello-oligomers, with cellotetraose being preferentially released. Similar proportions of soluble and insoluble reducing ends generated by hydrolysis of RAC indicated non-processive activity. Our study extends our knowledge of the molecular mechanism of cellulose hydrolysis by GH9 family endoglucanases with industrial relevance.

scholarly journals Evolutionary variance analysis of the Glycoside Hydrolase Family 13: Structural evidence in classification and evolution

2017 ◽  
Author(s):  
Jose Sergio Hleap ◽  
Christian Blouin
Keyword(s):  
Glycoside Hydrolase ◽  
Structural Information ◽  
Industrial Applications ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Novel Method ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Glycoside Hydrolase Family 13 ◽  
Structural Shifts

AbstractGlycoside Hydrolase Family 13 (GH13) structures are responsible for the hydrolysis of starch into smaller carbohydrates. They important in industrial applications and evolutionary studies. This family has been thoroughly documented in the the Carbohydrate-Active enZYmes Database (CAZY), and divided into subfamilies based mainly in sequence information. Here we give structural evidence into GH13 classification and evolution using structural information. Here we proposed a novel method that is sensitive enough to identify miss-classifications, or to provide evidence for further partition that can be of interests to bio-engineers and evolutionary biologists. We also introduced a method to explore the relative importance of residues with respect to the overall deformation that it causes to the overall structure in an evolutionary time scale. We found that the GH13 family can be classified into three main structural groups. There is a hierarchical structure within these clusters that can be use to inform other classification schemes. We also found that by using structural information, subtle structural shifts can be identified and that can be missed in sequence/phylogeny-only based classifications. When each structural group is explored, we found that identifying the most structurally variable sites can lead to identification of functionally (both catalytically and structurally) important residues.

scholarly journals Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

2005 ◽  
Vol 388 (3) ◽  
pp. 949-957 ◽  
Author(s):  
Masashi KIYOHARA ◽  
Keishi SAKAGUCHI ◽  
Kuniko YAMAGUCHI ◽  
Toshiyoshi ARAKI ◽  
Takashi NAKAMURA ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Carbohydrate Binding Modules ◽  
Hydrolysis Of ◽  
Hydrolase Family

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

scholarly journals Cloning, Expression and Characterization of a Novel Cold-adapted β-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 469 ◽  
Author(s):  
Jingjing Sun ◽  
Congyu Yao ◽  
Wei Wang ◽  
Zhiwei Zhuang ◽  
Junzhong Liu ◽  
...  
Keyword(s):  
Deep Sea ◽  
Glycoside Hydrolase ◽  
Sea Water ◽  
Maximum Activity ◽  
Glycoside Hydrolase Family ◽  
Cold Adapted ◽  
Hydrolysis Of ◽  
Hydrolase Family ◽  
Substrate Hydrolysis

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted β-galactosidase. A novel β-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S−1 at 35 °C with 2-nitrophenyl-β-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 β-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

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