Purification and Characterization of Intracellular α-Glucuronidase from<i>Aspergillus niger</i>5–16

Uchida, Hiromi; Nanri, Tomoko; Kawabata, Yasuyuki; Kusakabe, Isao; Murakami, Kazuo

doi:10.1271/bbb.56.1608

Cited by 31 publications

(22 citation statements)

References 13 publications

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“…It is possible also that in fungal ␣-glucuronidases, the much longer extra polypeptide chains at their C termini (ϳ110 residues) completely prevent the formation of dimers of the types observed in either AguA or GlcA67A and stabilize the monomers in solution. Such an arrangement is consistent with the reported monomeric forms of the fungal ␣-glucuronidases obtained by biochemical studies (11,18,33). It is noted that whereas all known bacterial ␣-glucuronidases are intracellular or cell associated, the fungal ␣-glucuronidases are extracellular.…”

Section: Discussionsupporting

confidence: 90%

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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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Section: Discussionsupporting

confidence: 90%

“…It is interesting that many fungal ␣-glucuronidases were found to be monomeric proteins (11,18,33). Indeed, the residues found in the dimerization positions of both groups I and II enzymes are no longer conserved in the group III fungal ␣-glucuronidases, in correlation with their observed monomeric characteristics.…”

Section: Resultsmentioning

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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“…Furthermore, as P. cellulosa GlcA67A exhibits similar activities against substrates containing one, two, and three xylose residues, the enzyme is likely to interact only with the aglycone sugar that participates in the target glycosidic bond. The importance of the aglycone moiety is supported by the results of a previous study, which showed that the Clostridium stercorarium and Thermoanaerobacterium saccarolyticum ␣-glucuronidases did not hydrolyze 4-nitrophenyl-␣-D-glucuronide but cleaved 4-O-methyl-D-glucuronoxylooligosaccharides (4,24). Furthermore, all the substrates evaluated in this study contain the uronic acid decoration at the nonreducing end of the xylose polymer (as indicatred in the Megazyme catalogue).…”

supporting

confidence: 75%

The Membrane-Bound α-Glucuronidase from Pseudomonas cellulosa Hydrolyzes 4- O- Methyl- d -Glucuronoxylooligosaccharides but Not 4- O- Methyl- d -Glucuronoxylan

et al. 2002

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The microbial degradation of xylan is a key biological process. Hardwood 4-O-methyl-D-glucuronoxylans are extensively decorated with 4-O-methyl-D-glucuronic acid, which is cleaved from the polysaccharides by ␣-glucuronidases. In this report we describe the primary structures of the ␣-glucuronidase from Cellvibrio mixtus (C. mixtus GlcA67A) and the ␣-glucuronidase from Pseudomonas cellulosa (P. cellulosa GlcA67A) and characterize P. cellulosa GlcA67A. The primary structures of C. mixtus GlcA67A and P. cellulosa GlcA67A, which are 76% identical, exhibit similarities with ␣-glucuronidases in glycoside hydrolase family 67. The membrane-associated pseudomonad ␣-glucuronidase released 4-O-methyl-D-glucuronic acid from 4-O-methyl-D-glucuronoxylooligosaccharides but not from 4-O-methyl-D-glucuronoxylan. We propose that the role of the glucuronidase, in combination with cell-associated xylanases, is to hydrolyze decorated xylooligosaccharides, generated by extracellular hemicellulases, to xylose and 4-O-methyl-D-glucuronic acid, enabling the pseudomonad to preferentially utilize the sugars derived from these polymers.

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“…Finally, the bacterial enzymes are typically dimeric with subunit molecular masses of about 70 kDa while most of the fungal enzymes are monomers with molecular masses from 100 to 160 kDa (e.g. Ishihara et al, 1990;Khandke et al, 1989;Uchida et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The K m of the ␣-glucuronidase of Thermoanaerobacterium sp. strain JW/SL-YS485 towards MeGlcAX 2 was 0.76 mM at 60ЊC (Shao et al, 1995), while the A. niger enzyme existed as two forms with K m values of 0.37 and 0.47 mM and V max values of 1.42 and 4.68 U mg ¹1 protein (at 60ЊC with MeGlcAX 3 as the substrate; Uchida et al, 1992). The partially purified ␣-glucuronidase of T. aurantiacus had a K m of 0.145 mM and a V max of 2.5 U mg ¹1 protein towards MeGlcAX 3 at 65ЊC (Khandke et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

Isolation and analysis of a gene encoding α‐glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan

Ruile

Winterhalter

Liebl

1997

Molecular Microbiology

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SummaryThis is the first report describing the analysis of a gene encoding an ␣-glucuronidase, an enzyme essential for the complete breakdown of substituted xylans. A DNA fragment that carries the gene for ␣-glucuronidase was isolated from chromosomal DNA of the hyperthermophilic bacterium Thermotoga maritima MSB8. The ␣-glucuronidase gene (aguA) was identified and characterized with the aid of nucleotide sequence analysis, deletion experiments and expression studies in Escherichia coli, and the start of the coding region was defined by amino-terminal sequencing of the purified recombinant enzyme. The aguA gene encodes a 674-amino-acid, largely hydrophilic polypeptide with a calculated molecular mass of 78 593 Da. The ␣-glucuronidase of T. maritima has a novel primary structure with no significant similarity to any other known amino acid sequence. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. Gel filtration analysis at low salt concentrations revealed a high apparent molecular mass (> 630 kDa) for the recombinant enzyme, but the oligomeric structure changed upon variation of the ionic strength or the pH, yielding hexameric and/or dimeric forms which were also enzymatically active. The enzyme hydrolysed 2-O-(4-O-methyl-␣-D-glucopyranosyluronic acid)-D-xylobiose (MeGlcAX 2 ) to xylobiose and 4-O-methylglucuronic acid. The K m for MeGlcAX 2 was 0.95 mM. The pH optimum was 6.3. Maximum activity was measured at 85ЊC, about 25ЊC or more above the values reported for all other ␣-glucuronidases known to date. When incubated at 55-75ЊC, the enzyme suffered partial inactivation, but thereafter the residual activity remained nearly constant for several days.

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Purification and Characterization of Intracellular α-Glucuronidase fromAspergillus niger5–16

Cited by 31 publications

References 13 publications

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

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

The Membrane-Bound α-Glucuronidase from Pseudomonas cellulosa Hydrolyzes 4- O- Methyl- d -Glucuronoxylooligosaccharides but Not 4- O- Methyl- d -Glucuronoxylan

Isolation and analysis of a gene encoding α‐glucuronidase, an enzyme with a novel primary structure involved in the breakdown of xylan

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