Crystal Structure of Mannanase 26A from Pseudomonas cellulosa and Analysis of Residues Involved in Substrate Binding

Hogg, Deborah; Woo, Eui-Jeon; Bolam, David N.; McKie, Vincent A.; Gilbert, Harry J.; Pickersgill, Richard W.

doi:10.1074/jbc.m010290200

Cited by 83 publications

(120 citation statements)

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“…The structure of CjMan26C ( Fig. 2A) shows a high degree of similarity with GH26 endo-␤1,4-mannanases, most notably with CjMan26A (27,29). Overlaying the structures of the two Cellvibrio enzymes reveals a root mean square deviation of 1.46 Å for 311 equivalent C ␣ atoms using SSM (30).…”

Section: ϫ2mentioning

confidence: 99%

“…These data show that the enzyme contains four subsites that extend from Ϫ2 to ϩ2, with bond cleavage occurring between the sugars located at Ϫ1 and ϩ1, by definition (26). CjMan26C is an extremely efficient enzyme, with a k cat /K m against mannotetraose ϳ20-fold higher than the catalytic efficiency of CjMan26A (27). Indeed, such a k cat /K m approaches catalytic perfection in which catalysis is limited by the diffusion rate (10…”

Section: ϫ2mentioning

confidence: 99%

“…4). These aromatic residues are highly conserved in GH26 enzymes exemplified by CjMan26A, where the structural equivalents to Trp-226 and Trp-373 are Trp-217 and Trp-360, respectively (27).…”

Section: Insights Into Substrate Binding Distortion and Catalysis Rmentioning

confidence: 99%

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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: ϫ2mentioning

confidence: 99%

Section: ϫ2mentioning

confidence: 99%

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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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“…Screening genomic databases for other GH26 enzymes that retain this Arg may facilitate the identification of novel mannooligosaccharidases. Currently, the two Cellvibrio enzymes that contain a highaffinity 22 subsite do not possess additional negative binding subsites, which may explain why the high activity displayed against mannotriose and mannotetraose is not translated to the hydrolysis of polysaccharides (Hogg et al, 2001;Cartmell et al, 2008).…”

Section: Xyloglucanmentioning

confidence: 99%

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

2010

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“…In recent years, crystallographic resolution of the structures of these enzymes has yielded information on their structure. At present, the tertiary structures of seven mannanases have been determined, of which five are from GH family 5 (12)(13)(14)(15)(16) and two are from family 26 (17,18). All of these mannanases share a common (␤/␣) 8 barrel fold, a retaining reaction mechanism (Fig.…”

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confidence: 99%

Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

Zhang

Peng

et al. 2008

Journal of Biological Chemistry

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An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-␤-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 Å resolution showed that AaManA adopted a (␤/␣) 8 -barrel fold. Two catalytic residues were identified: Glu 151 at the C terminus of ␤-stand ␤4 and Glu 231 at the C terminus of ␤7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr 95 and Cys 150 . We propose a model of substrate binding in AaManA in which Glu 282 interacts with the axial OH-C(2) in ؊2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.Mannans are polysaccharides found in plants and consist of a backbone of ␤-1,4-linked mannose and glucose units. Mannose residues often carry an ␣-galactosyl substitute at O-6, and the degree of substitution depends on the plant of origin (Fig. 1A) (1). Mannans are widely distributed in nature in parts of the hemicellulose fraction in hardwoods and softwoods (1), legume seeds (2), and beans of carob trees (3).Endo-␤-1,4-mannanases (mannanases; EC 3.2.1.78) are glycoside hydrolases that randomly cleave the ␤-1,4-linkage in mannans (Fig. 1, A and B) (4); these enzymes have been isolated from bacteria, fungi, plants, and some mollusks (5-8). Interest in these enzymes has been increasing due to their importance in hemicellulose hydrolysis and various other applications (5, 9, 10). During the last 2 decades, more than 80 sequences of the catalytic domains of mannanase have been reported (see the CAZy site on the World Wide Web) and classified into glycoside hydrolase (GH) 2 families 5 and 26, based on their sequence similarities (11). In recent years, crystallographic resolution of the structures of these enzymes has yielded information on their structure. At present, the tertiary structures of seven mannanases have been determined, of which five are from GH family 5 (12-16) and two are from family 26 (17, 18). All of these mannanases share a common (␤/␣) 8 barrel fold, a retaining reaction mechanism (Fig. 1C), and two conserved catalytic residues (two glutamate residues at the C termini of ␤4 and ␤7, respectively). Therefore, all of these enzymes have been assigned to the same GH ...

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Crystal Structure of Mannanase 26A from Pseudomonas cellulosa and Analysis of Residues Involved in Substrate Binding

Cited by 83 publications

References 33 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

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

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