Subsite Structure of Saccharomycopsis α-Amylase Secreted from Saccharomyces cerevisiae

In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-␤(1,4)-glucanase or ␤(1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme from Saccharophagus degradans (SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides. SdGluc5_26A displays lichenase activity toward ␤(1,3; 1,4)-glucans with a side cellobiohydrolase activity toward ␤(1, 4)-glucans. The three-dimensional structure of SdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region of SdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization of SdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity of SdGluc5_26A. Its deletion opens the enzyme cleft at the ؊3 subsite and turns the enzyme into an endo-␤(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.

Section: Methodsmentioning

confidence: 99%

The Quaternary Structure of a Glycoside Hydrolase Dictates Specificity toward β-Glucans

Lafond

Sulzenbacher

Freyd

et al. 2016

“…The protocol used is the same as described by Cervera Tison et al (33), except that the A buffer was composed of 5 mM NaOAc and 80 mM NaOH. To evaluate the activity of the AgaSK on ␣-galactosides (melibiose and raffinose), the initial slopes of progress curves were used to determine the catalytic efficiency (k cat /K m ) of the reaction, following the equation of Matsui et al (34). All assays were carried out in triplicate.…”

Section: Methodsmentioning

confidence: 99%

α-Galactosidase/Sucrose Kinase (AgaSK), a Novel Bifunctional Enzyme from the Human Microbiome Coupling Galactosidase and Kinase Activities

Bruel

Sulzenbacher

Tison³

et al. 2011

Background:Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes. Results: AgaSK is a protein coupling galactosidase and sucrose kinase activity. The structure of the galactosidase domain sheds light onto substrate recognition. Conclusion: AgaSK produces sucrose-6-phosphate directly from raffinose. Significance: Production of sucrose-6-phosphate directly from raffinose points toward a novel glycolytic pathway in bacteria.

“…Each assay was performed in triplicate. To evaluate the activity of the two xylanases against xylooligosaccharides, progress curves were carried out as described previously, and the data were used to determine k cat /K m following the equation of Matsui et al (25). The bond cleavage frequency and k cat /K m data obtained from these experiments were used to calculate the ⌬G of xylose binding at each of the subsites following the method of Suganuma et al (26).…”

Section: Methodsmentioning

confidence: 99%

The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

Pell

Taylor

Gloster

et al. 2004

165

136

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.