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

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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Crystal structure of inactivated Thermotoga maritima invertase in complex with the trisaccharide substrate raffinose

Biochemical Journal ◽

10.1042/bj20051936 ◽

2006 ◽

Vol 395 (3) ◽

pp. 457-462 ◽

Cited By ~ 49

Author(s):

François Alberto ◽

Emmanuelle Jordi ◽

Bernard Henrissat ◽

Mirjam Czjzek

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Thermotoga Maritima ◽

Three Dimensional ◽

Catalytic Efficiency ◽

Dimensional Structure ◽

Glycoside Hydrolase Family ◽

Acid Base ◽

Hydrolase Family ◽

Binding Subsites

Thermotoga maritima invertase (β-fructosidase), a member of the glycoside hydrolase family GH-32, readily releases β-D-fructose from sucrose, raffinose and fructan polymers such as inulin. These carbohydrates represent major carbon and energy sources for prokaryotes and eukaryotes. The invertase cleaves β-fructopyranosidic linkages by a double-displacement mechanism, which involves a nucleophilic aspartate and a catalytic glutamic acid acting as a general acid/base. The three-dimensional structure of invertase shows a bimodular enzyme with a five bladed β-propeller catalytic domain linked to a β-sandwich of unknown function. In the present study we report the crystal structure of the inactivated invertase in interaction with the natural substrate molecule α-D-galactopyranosyl-(1,6)-α-D-glucopyranosyl-β-D-fructofuranoside (raffinose) at 1.87 Å (1 Å=0.1 nm) resolution. The structural analysis of the complex reveals the presence of three binding-subsites, which explains why T. maritima invertase exhibits a higher affinity for raffinose than sucrose, but a lower catalytic efficiency with raffinose as substrate than with sucrose.

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GH30-7 Endoxylanase C from the Filamentous Fungus Talaromyces cellulolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.01442-19 ◽

2019 ◽

Vol 85 (22) ◽

Author(s):

Yusuke Nakamichi ◽

Tatsuya Fujii ◽

Thierry Fouquet ◽

Akinori Matsushika ◽

Hiroyuki Inoue

Keyword(s):

Glycoside Hydrolase ◽

Glucuronic Acid ◽

Catalytic Domain ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Promising Tool ◽

Talaromyces Cellulolyticus ◽

Food And Feed ◽

Cellulose Binding ◽

Hydrolysis Of

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 Cel44A, a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium thermocellum

Journal of Biological Chemistry ◽

10.1074/jbc.m706835200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 35703-35711 ◽

Cited By ~ 35

Author(s):

Yu Kitago ◽

Shuichi Karita ◽

Nobuhisa Watanabe ◽

Masakatsu Kamiya ◽

Tomoyasu Aizawa ◽

...

Keyword(s):

Crystal Structure ◽

Glutamic Acid ◽

Glycoside Hydrolase ◽

Clostridium Thermocellum ◽

Structural Features ◽

Glycoside Hydrolase Family ◽

Wild Type ◽

H Nmr ◽

Hydrolase Family

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96Å. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a β-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu186 and Glu359, are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a 1H NMR experiment using the reduced cellooligosaccharide as a substrate.

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Structural and functional characterization of a glycoside hydrolase family 3 β-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7

The Journal of Biochemistry ◽

10.1093/jb/mvz072 ◽

2019 ◽

Vol 166 (6) ◽

pp. 503-515

Author(s):

Takafumi Itoh ◽

Tomomitsu Araki ◽

Tomohiro Nishiyama ◽

Takao Hibi ◽

Hisashi Kimoto

Keyword(s):

Crystal Structure ◽

Glycoside Hydrolase ◽

Catalytic Mechanism ◽

Functional Characterization ◽

Structural Features ◽

Expression Cloning ◽

Glycoside Hydrolase Family ◽

Paenibacillus Sp ◽

Hydrolase Family

Abstract Chitin, a β-1,4-linked homopolysaccharide of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N′-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus β-N-acetyl-d-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2–4, 4-nitrophenyl N-acetyl β-d-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2–6, but has no detectable activity against 4-nitrophenyl β-d-glucose, 4-nitrophenyl β-d-galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 β-N-acetyl-d-glucosaminidase from Paenibacillus sp.

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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Crystal structure of the Enterococcus faecalis α‐ N ‐acetylgalactosaminidase, a member of the glycoside hydrolase family 31

FEBS Letters ◽

10.1002/1873-3468.13804 ◽

2020 ◽

Vol 594 (14) ◽

pp. 2282-2293

Author(s):

Takatsugu Miyazaki ◽

Enoch Y. Park

Keyword(s):

Crystal Structure ◽

Enterococcus Faecalis ◽

Glycoside Hydrolase ◽

Glycoside Hydrolase Family ◽

Glycoside Hydrolase Family 31 ◽

Hydrolase Family

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A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.001213 ◽

2018 ◽

Vol 293 (21) ◽

pp. 7993-8008 ◽

Cited By ~ 8

Author(s):

Subrata Debnath ◽

Dalibor Kosek ◽

Harichandra D. Tagad ◽

Stewart R. Durell ◽

Daniel H. Appella ◽

...

Keyword(s):

Crystal Structure ◽

Metal Ions ◽

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Phosphatases ◽

Random Order ◽

Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

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