A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates

Hägglund, Per; Eriksson, Torny; Collén, Anna; Nerinckx, Wim; Claeyssens, Marc; Stålbrand, Henrik

doi:10.1016/s0168-1656(02)00290-0

Cited by 92 publications

(67 citation statements)

References 44 publications

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“…By contrast, endo-acting GH5 mannanases often contain cellulose-targeted CBMs (8,43,44), and thus the function of GH5 and GH26 C. japonicus mannanases proposed here is likely to have a generic biolog-FIGURE 6. The mannanase repertoire of C. japonicus.…”

Section: Discussionmentioning

confidence: 96%

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

Cartmell

Topakas

Ducros

et al. 2008

Journal of Biological Chemistry

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The microbial degradation of the plant cell wall is a pivotal biological process that is of increasing industrial significance. One of the major plant structural polysaccharides is mannan, a ␤-1,4-linked D-mannose polymer, which is hydrolyzed by endoand exo-acting mannanases. The mechanisms by which the exoacting enzymes target the chain ends of mannan and how galactose decorations influence activity are poorly understood. Here we report the crystal structure and biochemical properties of CjMan26C, a Cellvibrio japonicus GH26 mannanase. The exoacting enzyme releases the disaccharide mannobiose from the nonreducing end of mannan and mannooligosaccharides, harnessing four mannose-binding subsites extending from ؊2 to ؉2. The structure of CjMan26C is very similar to that of the endo-acting C. japonicus mannanase CjMan26A. The exo-activity displayed by CjMan26C, however, reflects a subtle change in surface topography in which a four-residue extension of surface loop creates a steric block at the distal glycone ؊2 subsite. endoActivity can be introduced into enzyme variants through truncation of an aspartate side chain, a component of a surface loop, or by removing both the aspartate and its flanking residues. The structure of catalytically competent CjMan26C, in complex with a decorated manno-oligosaccharide, reveals a predominantly unhydrolyzed substrate in an approximate 1 S 5 conformation. The complex structure helps to explain how the substrate "side chain" decorations greatly reduce the activity of the enzyme; the galactose side chain at the ؊1 subsite makes polar interactions with the aglycone mannose, possibly leading to suboptimal binding and impaired leaving group departure. This report reveals how subtle differences in the loops surrounding the active site of a glycoside hydrolase can lead to a change in the mode of action of the enzyme.The plant cell wall represents the dominant source of organic carbon in the biosphere and as such supports many facets of terrestrial and marine life. Access to this valuable source of carbon is mediated by extensive microbial enzyme consortia, which generate sugars that are utilized by microbial ecosystems to the benefit of plants, mammalian herbivores, and insects. These degradative enzymes are increasingly deployed in industrial processes, and it is apparent that their use in producing second generation, lignocellulosebased, biofuels is of considerable environmental significance (1, 2). Among the major polysaccharides in softwoods and angiosperms are the mannans. These polysaccharides comprise a backbone of ␤-1,4-linked D-mannopyranose sugars (known as "mannan") or a heterogeneous combination of ␤-1,4-D-mannose and ␤-1,4-D-glucose units (termed "glucomannan"), which can be decorated with ␣-1,6-galactose side chains to yield "galactomannan" and "galactoglucomannan," respectively (3). For complete enzymatic degradation, the galactose decorations are removed by ␣-galactosidases (4, 5), whereas the internal glycosidic linkages in mannans are cleaved by endo-␤-1,4 mannanases ...

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Section: Discussionmentioning

confidence: 96%

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

Cartmell

Topakas

Ducros

et al. 2008

Journal of Biological Chemistry

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“…Several ␤-mannanases produced by terrestrial bacteria and fungi found in environments containing decaying plant material have been characterized in molecular detail (5). For example, the mode of action and the crystal structure have been determined for the GH5 ␤-mannanases of the bacterium Thermomonospora fusca (9) and the fungus Trichoderma reesei (10)(11)(12), the GH26 ␤-mannanase CfMan26A from the bacterium Cellulomonas fimi (13)(14)(15)(16), and the GH5 and GH26 ␤-mannanases of the bacterium Cellvibrio japonicus (17,18). Also, the crystal structures of GH26 ␤-mannanases from Bacillus subtilis strains and from the thermophilic bacterium Alicyclobacillus acidocaldarius (GH113) have been described previously (19)(20)(21).…”

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“…The hydrolysis products were quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex, Sunnyvale, CA), as described by Hägglund et al (10). A Carbopac PA-100 column (Dionex) was used for the separation with an isocratic flow of 78 mM NaOH.…”

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“…The gels were stained as described above. Binding toward insoluble polysaccharides was performed as described by Häg-glund et al (10). Washed ivory nut mannan and Avicel cellulose (Avicel PH-101; Fluka, Ireland) were suspended to a concentration of 10 g/liter in 100 mM Na phosphate-50 mM Na citrate buffer at pH 7.0, with 0.2 M enzyme added to the tubes, which were then incubated at 4°C for 1 h with continued stirring.…”

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Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

Kulcinskaja

Rosengren

Ibrahim

et al. 2013

Appl Environ Microbiol

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The gene encoding ␤-mannanase (EC 3.2.1.78) BaMan26A from the bacterium Bifidobacterium adolescentis (living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed in Escherichia coli without the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 M (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K, k cat was determined to be 444 s ؊1 and K m 21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.

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“…The CBD in some cell wall-degrading enzymes, such as endo-1,4-␤-glucanase and -␤-mannanase, critically serves in the hydrolysis of their responsible substrates (i.e., substrates the enzymes can hydrolyze), whereas the truncated structure (catalytic core domain only) has reduced activity and the addition of another CBD can enhance activity (16,17,26). Similarly, the hydrolytic activity of MoCel6A on cellulose was increased by a tandemly aligned CBD (CBD-MoCel6A) and decreased with the removal of the CBD [MoCel6A(cd)] (Fig.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

Takahashi

Nakano

et al. 2010

Appl Environ Microbiol

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Three GH-6 family cellobiohydrolases are expected in the genome of Magnaporthe grisea based on the complete genome sequence. Here, we demonstrate the properties, kinetics, and substrate specificities of a Magnaporthe oryzae GH-6 family cellobiohydrolase (MoCel6A). In addition, the effect of cellobiose on MoCel6A activity was also investigated. MoCel6A contiguously fused to a histidine tag was overexpressed in M. oryzae and purified by affinity chromatography. MoCel6A showed higher hydrolytic activities on phosphoric acidswollen cellulose (PSC), ␤-glucan, and cellooligosaccharide derivatives than on cellulose, of which the best substrates were cellooligosaccharides. A tandemly aligned cellulose binding domain (CBD) at the N terminus caused increased activity on cellulose and PSC, whereas deletion of the CBD (catalytic domain only) showed decreased activity on cellulose. MoCel6A hydrolysis of cellooligosaccharides and sulforhodamine-conjugated cellooligosaccharides was not inhibited by exogenously adding cellobiose up to 438 mM, which, rather, enhanced activity, whereas a GH-7 family cellobiohydrolase from M. oryzae (MoCel7A) was severely inhibited by more than 29 mM cellobiose. Furthermore, we assessed the effects of cellobiose on hydrolytic activities using MoCel6A and Trichoderma reesei cellobiohydrolase (TrCel6A), which were prepared in Aspergillus oryzae. MoCel6A showed increased hydrolysis of cellopentaose used as a substrate in the presence of 292 mM cellobiose at pH 4.5 and pH 6.0, and enhanced activity disappeared at pH 9.0. In contrast, TrCel6A exhibited slightly increased hydrolysis at pH 4.5, and hydrolysis was severely inhibited at pH 9.0. These results suggest that enhancement or inhibition of hydrolytic activities by cellobiose is dependent on the reaction mixture pH.

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A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates

Cited by 92 publications

References 44 publications

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

Characterization of a Cellobiohydrolase (MoCel6A) Produced by Magnaporthe oryzae

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