The α-Glucuronidase, GlcA67A, of Cellvibrio japonicus Utilizes the Carboxylate and Methyl Groups of Aldobiouronic Acid as Important Substrate Recognition Determinants

Nagy, Tibor; Nurizzo, Didier; Davies, G.J.; Biely, Peter; Lakey, Jeremy H.; Bolam, David N.; Gilbert, Harry J.

doi:10.1074/jbc.m302205200

Cited by 33 publications

(27 citation statements)

References 22 publications

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“…The hydroxyl group of Tyr-200 and the OD-2 of Asp-161 make hydrogen bonds with the O-2 of 4-OMeGlcA, and the pyranose sugar ring in the ϩ1 subsite makes hydrophobic interactions with the aromatic ring of Phe-340. In contrast, studies reported in the accompanying paper show that when MX 3 and MX 2 are bound in the aglycone region of the substrate binding cleft of SoXyn10A, the electron density for the 4-O-MeGlcA is not apparent, suggesting that the side chain is disordered (19). The difference between CmXyn10B and SoXyn10A may reflect generally weaker binding of the decorated substrate to the distal aglycone binding sites of the Streptomyces enzyme, as evidenced by the absence of a xylose moiety at the ϩ3 subsite.…”

Section: Figcontrasting

confidence: 39%

“…The arabinofuranoside moiety is in one discrete ordered conformation and does not interact directly with the protein but hydrogen bonds both to solvent and through a 2.7-Å hydrogen bond from O-3 to the endocyclic O-5 of the Ϫ3 subsite xyloside. Xylotriose bound at subsites ϩ1 to ϩ3 subsites may be a trace contaminant; AX 3 would not be able to bind at these subsites, as steric clashes would prevent decoration of the O-3 of xylose at the ϩ2 subsite (Fig. 5c).…”

Section: Cmxyn10b-mxmentioning

confidence: 99%

“…This figure, shown in divergent (wall-eyed) stereo, was drawn with BOBSCRIPT (35). 3 Complex-The structure of CmXyn10B E262S in complex with MX 3 (Fig. 5a) was refined to a final R-factor of 0.16 and R free of 0.20.…”

Section: Figmentioning

confidence: 99%

“…The extent and nature of such decoration varies between plant species. For complete degradation the arabinofuranosyl and acetyl side chains are removed from the polysaccharide by arabinofuranosidases and acetyl xylan esterases, respectively (1), whereas the ␣-glucuronidases, which release 4-O-MeGlcA, act only on xylooligosaccharides in which the xylose at the nonreducing end is decorated with the uronic acid (2,3).…”

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confidence: 99%

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The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

Pell

Taylor

Gloster

et al. 2004

Journal of Biological Chemistry

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165

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Endo-␤-1,4-xylanases (xylanases), which cleave ␤-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the threedimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the ؉1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the ؉4, ؉3, ؉1, and ؊3 subsites may allow accommodation of the ␣-1,2-linked 4-O-methyl-D-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the ؉1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3-linked L-arabinofuranoside decorations is observed in the ؊2 subsite and could, most likely, be tolerated when bound to xylosides in ؊3 and ؉4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as ␣-glucuronidase. The data described in this report and in the accompanying paper (Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2004) J. Biol. Chem. 279, 9606 -9614) indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases.

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Section: Figcontrasting

confidence: 39%

Section: Cmxyn10b-mxmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

Pell

Taylor

Gloster

et al. 2004

Journal of Biological Chemistry

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165

136

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“…The enzymes of this family were found to use a single displacement-inverting mechanism for the hydrolysis of the glycosidic bond (3). Currently, complete three-dimensional structures of only two representatives of GH-67 enzymes are available, the ␣-glucuronidase from Cellvibrio japonicus (formerly Pseudomonas cellulosa), GlcA67A (22,23), and the ␣-glucuronidase from Geobacillus stearothermophilus, AguA (12). The overall structures of these two enzymes appear to be quite similar.…”

mentioning

confidence: 99%

Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase fromGeobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

et al. 2004

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The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. ␣-Glucuronidases are family 67 glycosidases that cleave the ␣-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of ␣-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the ␣-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial ␣-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in ␣-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.

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Enzymatic Hydrolysis of Lignocellulosic Biomass

Stickel

Elander

McMillan

et al. 2014

Bioprocessing of Renewable Resources to Commodity Bioproducts

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The α-Glucuronidase, GlcA67A, of Cellvibrio japonicus Utilizes the Carboxylate and Methyl Groups of Aldobiouronic Acid as Important Substrate Recognition Determinants

Cited by 33 publications

References 22 publications

The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase fromGeobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

Enzymatic Hydrolysis of Lignocellulosic Biomass

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