GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

ABSTRACT Glycoside hydrolase family 30 subfamily 7 (GH30-7) enzymes include various types of xylanases, such as glucuronoxylanase, endoxylanase, xylobiohydrolase, and reducing-end xylose-releasing exoxylanase. Here, we characterized the mode of action and gene expression of the GH30-7 endoxylanase from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C). TcXyn30C has a modular structure consisting of a GH30-7 catalytic domain and a C-terminal cellulose binding module 1, whose cellulose-binding ability has been confirmed. Sequence alignment of GH30-7 xylanases exhibited that TcXyn30C has a conserved Phe residue at the position corresponding to a conserved Arg residue in GH30-7 glucuronoxylanases, which is required for the recognition of the 4-O-methyl-α-d-glucuronic acid (MeGlcA) substituent. TcXyn30C degraded both glucuronoxylan and arabinoxylan with similar kinetic constants and mainly produced linear xylooligosaccharides (XOSs) with 2 to 3 degrees of polymerization, in an endo manner. Notably, the hydrolysis of glucuronoxylan caused an accumulation of 22-(MeGlcA)-xylobiose (U4m2X). The production of this acidic XOS is likely to proceed via multistep reactions by putative glucuronoxylanase activity that produces 22-(MeGlcA)-XOSs (XnU4m2X, n ≥ 0) in the initial stages of the hydrolysis and by specific release of U4m2X from a mixture containing XnU4m2X. Our results suggest that the unique endoxylanase activity of TcXyn30C may be applicable to the production of linear and acidic XOSs. The gene xyn30C was located adjacent to the putative GH62 arabinofuranosidase gene (abf62C) in the T. cellulolyticus genome. The expression of both genes was induced by cellulose. The results suggest that TcXyn30C may be involved in xylan removal in the hydrolysis of lignocellulose by the T. cellulolyticus cellulolytic system. IMPORTANCE Xylooligosaccharides (XOSs), which are composed of xylose units with a β-1,4 linkage, have recently gained interest as prebiotics in the food and feed industry. Apart from linear XOSs, branched XOSs decorated with a substituent such as methyl glucuronic acid and arabinose also have potential applications. Endoxylanase is a promising tool in producing XOSs from xylan. The structural variety of XOSs generated depends on the substrate specificity of the enzyme as well as the distribution of the substituents in xylan. Thus, the exploration of endoxylanases with novel specificities is expected to be useful in the provision of a series of XOSs. In this study, the endoxylanase TcXyn30C from Talaromyces cellulolyticus was characterized as a unique glycoside hydrolase belonging to the family GH30-7, which specifically releases 22-(4-O-methyl-α-d-glucuronosyl)-xylobiose from hardwood xylan. This study provides new insights into the production of linear and branched XOSs by GH30-7 endoxylanase.

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Crystal structure of GH30-7 endoxylanase C from the filamentous fungus Talaromyces cellulolyticus

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20009024 ◽

2020 ◽

Vol 76 (8) ◽

pp. 341-349

Author(s):

Yusuke Nakamichi ◽

Tatsuya Fujii ◽

Masahiro Watanabe ◽

Akinori Matsushika ◽

Hiroyuki Inoue

Keyword(s):

Crystal Structure ◽

Glucuronic Acid ◽

Catalytic Domain ◽

Catalytic Efficiency ◽

Structural Features ◽

Hydroxyl Group ◽

Glycoside Hydrolase Family ◽

Talaromyces Cellulolyticus ◽

Cellulose Binding ◽

Hydrolase Family

GH30-7 endoxylanase C from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C) belongs to glycoside hydrolase family 30 subfamily 7, and specifically releases 22-(4-O-methyl-α-D-glucuronosyl)-xylobiose from glucuronoxylan, as well as various arabino-xylooligosaccharides from arabinoxylan. TcXyn30C has a modular structure consisting of a catalytic domain and a C-terminal cellulose-binding module 1 (CBM1). In this study, the crystal structure of a TcXyn30C mutant which lacks the CBM1 domain was determined at 1.65 Å resolution. The structure of the active site of TcXyn30C was compared with that of the bifunctional GH30-7 xylanase B from T. cellulolyticus (TcXyn30B), which exhibits glucuronoxylanase and xylobiohydrolase activities. The results revealed that TcXyn30C has a conserved structural feature for recognizing the 4-O-methyl-α-D-glucuronic acid (MeGlcA) substituent in subsite −2b. Additionally, the results demonstrated that Phe47 contributes significantly to catalysis by TcXyn30C. Phe47 is located in subsite −2b and also near the C-3 hydroxyl group of a xylose residue in subsite −2a. Substitution of Phe47 with an arginine residue caused a remarkable decrease in the catalytic efficiency towards arabinoxylan, suggesting the importance of Phe47 in arabinoxylan hydrolysis. These findings indicate that subsite −2b of TcXyn30C has unique structural features that interact with arabinofuranose and MeGlcA substituents.

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Mode of Action of GH30-7 Reducing-End Xylose-Releasing Exoxylanase A (Xyn30A) from the Filamentous Fungus Talaromyces cellulolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.00552-19 ◽

2019 ◽

Vol 85 (13) ◽

Cited By ~ 3

Author(s):

Yusuke Nakamichi ◽

Thierry Fouquet ◽

Shotaro Ito ◽

Akinori Matsushika ◽

Hiroyuki Inoue

Keyword(s):

Mode Of Action ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Fungal Enzymes ◽

Content Type ◽

Talaromyces Cellulolyticus ◽

Bacteria And Fungi ◽

Hydrolysis Of ◽

Hydrolase Family ◽

Glycoside Hydrolase Family 30

ABSTRACT In this study, we characterized the mode of action of reducing-end xylose-releasing exoxylanase (Rex), which belongs to the glycoside hydrolase family 30-7 (GH30-7). GH30-7 Rex, isolated from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), exists as a dimer. The purified Xyn30A released xylose from linear xylooligosaccharides (XOSs) 3 to 6 xylose units in length with similar kinetic constants. Hydrolysis of branched, borohydride-reduced, and p-nitrophenyl XOSs clarified that Xyn30A possesses a Rex activity. 1H nuclear magnetic resonance (1H NMR) analysis of xylotriose hydrolysate indicated that Xyn30A degraded XOSs via a retaining mechanism and without recognizing an anomeric structure at the reducing end. Hydrolysis of xylan by Xyn30A revealed that the enzyme continuously liberated both xylose and two types of acidic XOSs: 22-(4-O-methyl-α-d-glucuronyl)-xylotriose (MeGlcA2Xyl3) and 22-(MeGlcA)-xylobiose (MeGlcA2Xyl2). These acidic products were also detected during hydrolysis using a mixture of MeGlcA2Xyln (n = 2 to 14) as the substrate. This indicates that Xyn30A can release MeGlcA2Xyln (n = 2 and 3) in an exo manner. Comparison of subsites in Xyn30A and GH30-7 glucuronoxylanase using homology modeling suggested that the binding of the reducing-end residue at subsite +2 was partially prevented by a Gln residue conserved in GH30-7 Rex; additionally, the Arg residue at subsite −2b, which is conserved in glucuronoxylanase, was not found in Xyn30A. Our results lead us to propose that GH30-7 Rex plays a complementary role in hydrolysis of xylan by fungal cellulolytic systems. IMPORTANCE Endo- and exo-type xylanases depolymerize xylan and play crucial roles in the assimilation of xylan in bacteria and fungi. Exoxylanases release xylose from the reducing or nonreducing ends of xylooligosaccharides; this is generated by the activity of endoxylanases. β-Xylosidase, which hydrolyzes xylose residues on the nonreducing end of a substrate, is well studied. However, the function of reducing-end xylose-releasing exoxylanases (Rex), especially in fungal cellulolytic systems, remains unclear. This study revealed the mode of xylan hydrolysis by Rex from the cellulolytic fungus Talaromyces cellulolyticus (Xyn30A), which belongs to the glycoside hydrolase family 30-7 (GH30-7). A conserved residue related to Rex activity is found in the substrate-binding site of Xyn30A. These findings will enhance our understanding of the function of GH30-7 Rex in the cooperative hydrolysis of xylan by fungal enzymes.

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Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation

Applied and Environmental Microbiology ◽

10.1128/aem.01744-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6380-6392 ◽

Cited By ~ 26

Author(s):

Manuel Nieto-Domínguez ◽

Laura I. de Eugenio ◽

Jorge Barriuso ◽

Alicia Prieto ◽

Beatriz Fernández de Toro ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Gene Encoding ◽

Intronless Gene ◽

Content Type ◽

Alkyl Glycosides ◽

Beechwood Xylan ◽

Dimeric Form ◽

Culture Supernatants ◽

Hydrolysis Of

ABSTRACTThis paper reports on a novel β-xylosidase from the hemicellulolytic fungusTalaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, andKmandVmaxvalues of 0.17 mM and 52.0 U/mg, respectively, using commercialp-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02582-18 ◽

2019 ◽

Vol 85 (6) ◽

Cited By ~ 8

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.

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A New Group of Modular Xylanases in Glycoside Hydrolase Family 8 from Marine Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.01785-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Xiu-Lan Chen ◽

Fang Zhao ◽

Yong-Sheng Yue ◽

Xi-Ying Zhang ◽

Yu-Zhong Zhang ◽

...

Keyword(s):

Marine Bacteria ◽

Glycoside Hydrolase ◽

Catalytic Domain ◽

Domain Architecture ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Binding Ability ◽

Content Type ◽

Hydrolase Family

ABSTRACT Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight β-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two α-helices, contains one of the α-helices of the catalytic domain's (α/α)6 barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (≥80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases. IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.

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Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

Applied and Environmental Microbiology ◽

10.1128/aem.02725-12 ◽

2012 ◽

Vol 79 (3) ◽

pp. 989-996 ◽

Cited By ~ 38

Author(s):

Fei Zheng ◽

Shaojun Ding

Keyword(s):

Glycoside Hydrolase ◽

Catalytic Domain ◽

Specific Activity ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Volvariella Volvacea ◽

Reaction Conditions ◽

Content Type ◽

C Terminus ◽

Hydrolase Family

ABSTRACTEG1 is a modular glycoside hydrolase family 5 endoglucanase fromVolvariella volvaceaconsisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses.

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Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

Eukaryotic Cell ◽

10.1128/ec.00187-10 ◽

2010 ◽

Vol 9 (11) ◽

pp. 1650-1660 ◽

Cited By ~ 11

Author(s):

Encarnación Dueñas-Santero ◽

Ana Belén Martín-Cuadrado ◽

Thierry Fontaine ◽

Jean-Paul Latgé ◽

Francisco del Rey ◽

...

Keyword(s):

Cell Wall ◽

Schizosaccharomyces Pombe ◽

Protein Characterization ◽

Wall Material ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Hydrolysis Of ◽

Controlled Hydrolysis ◽

Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

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A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40Tand Its Transglycosylase Activity

Applied and Environmental Microbiology ◽

10.1128/aem.00635-16 ◽

2016 ◽

Vol 82 (14) ◽

pp. 4340-4349 ◽

Cited By ~ 9

Author(s):

Damao Wang ◽

Do Hyoung Kim ◽

Nari Seo ◽

Eun Ju Yun ◽

Hyun Joo An ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Marine Bacterium ◽

Enzymatic Reaction ◽

Glycoside Hydrolase Family ◽

Reaction Products ◽

Brown Macroalgae ◽

Fungal Cell ◽

Content Type ◽

Saccharophagus Degradans ◽

Hydrolase Family

ABSTRACTIn this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. Thegly5Mgene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) inSaccharophagus degradans2-40T. Thegly5Mgene was cloned and overexpressed inEscherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization–tandem time of flight mass spectrometry, Gly5M was identified as a novel β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes.IMPORTANCEIn this study, we have discovered a novel β-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium,Saccharophagus degradans2-40T. Gly5M was identified as the newly found β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.

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Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function

Microbial Genomics ◽

10.1099/mgen.0.000404 ◽

2020 ◽

Vol 6 (10) ◽

Author(s):

Ao Li ◽

Elisabeth Laville ◽

Laurence Tarquis ◽

Vincent Lombard ◽

David Ropartz ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Sequence Similarity ◽

Gut Bacteria ◽

Glycoside Hydrolase Family ◽

Sequence Alignments ◽

Multiple Sequence ◽

Content Type ◽

Multiple Sequence Alignments ◽

Hydrolase Family

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target β-1,2-, β-1,3- and β-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known β-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

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

Scientific Reports ◽

10.1038/s41598-020-77133-8 ◽

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.

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