Abstract:Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK a values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine pr… Show more
“…7a,b). This particular conformation for Glu176/Tyr72 has been observed before in the neutron crystal structure of T. reesei Xyn11 at pD4–4.4 with an empty active site (PDB 4s2f), which was shown to be able to return to a catalytically competent conformation at basic pD values [40, 42]. Fig.…”
BackgroundReplacing fossil fuel with renewable sources such as lignocellulosic biomass is currently a promising alternative for obtaining biofuel and for fighting against the consequences of climate change. However, the recalcitrant structure of lignocellulosic biomass residues constitutes a major limitation for its widespread use in industry. The efficient hydrolysis of lignocellulosic materials requires the complementary action of multiple enzymes including xylanases and β-xylosidases, which are responsible for cleaving exo- and endoxylan linkages, that release oligocarbohydrates that can be further processed by other enzymes.ResultsWe have identified the endo-β-1,4-xylanase Xyl2 from Fusarium oxysporum as a promising glycoside hydrolase family 11 enzyme for the industrial degradation of xylan. To characterize Xyl2, we have cloned the synthetic optimized gene and expressed and purified recombinant Xyl2 to homogeneity, finally obtaining 10 mg pure Xyl2 per liter of culture. The crystal structure of Xyl2 at 1.56 Å resolution and the structure of a methyl-xylopyranoside Xyl2 complex at 2.84 Å resolution cast a highly detailed view of the active site of the enzyme, revealing the molecular basis for the high catalytic efficiency of Xyl2. The kinetic analysis of Xyl2 demonstrates high xylanase activity and non-negligible β-xylosidase activity under a variety of experimental conditions including alkaline pH and elevated temperature. Immobilizing Xyl2 on a variety of solid supports enhances the enzymatic properties that render Xyl2 a promising industrial biocatalyst, which, together with the detailed structural data, may establish Xyl2 as a platform for future developments of industrially relevant xylanases.Conclusions
F. oxysporum Xyl2 is a GH11 xylanase which is highly active in free form and immobilized onto a variety of solid supports in a wide pH range. Furthermore, immobilization of Xyl2 on certain supports significantly increases its thermal stability. A mechanistic rationale for Xyl2's remarkable catalytic efficiency at alkaline pH is proposed on the basis of two crystallographic structures. Together, these properties render Xyl2 an attractive biocatalyst for the sustainable industrial degradation of xylan.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0605-z) contains supplementary material, which is available to authorized users.
“…7a,b). This particular conformation for Glu176/Tyr72 has been observed before in the neutron crystal structure of T. reesei Xyn11 at pD4–4.4 with an empty active site (PDB 4s2f), which was shown to be able to return to a catalytically competent conformation at basic pD values [40, 42]. Fig.…”
BackgroundReplacing fossil fuel with renewable sources such as lignocellulosic biomass is currently a promising alternative for obtaining biofuel and for fighting against the consequences of climate change. However, the recalcitrant structure of lignocellulosic biomass residues constitutes a major limitation for its widespread use in industry. The efficient hydrolysis of lignocellulosic materials requires the complementary action of multiple enzymes including xylanases and β-xylosidases, which are responsible for cleaving exo- and endoxylan linkages, that release oligocarbohydrates that can be further processed by other enzymes.ResultsWe have identified the endo-β-1,4-xylanase Xyl2 from Fusarium oxysporum as a promising glycoside hydrolase family 11 enzyme for the industrial degradation of xylan. To characterize Xyl2, we have cloned the synthetic optimized gene and expressed and purified recombinant Xyl2 to homogeneity, finally obtaining 10 mg pure Xyl2 per liter of culture. The crystal structure of Xyl2 at 1.56 Å resolution and the structure of a methyl-xylopyranoside Xyl2 complex at 2.84 Å resolution cast a highly detailed view of the active site of the enzyme, revealing the molecular basis for the high catalytic efficiency of Xyl2. The kinetic analysis of Xyl2 demonstrates high xylanase activity and non-negligible β-xylosidase activity under a variety of experimental conditions including alkaline pH and elevated temperature. Immobilizing Xyl2 on a variety of solid supports enhances the enzymatic properties that render Xyl2 a promising industrial biocatalyst, which, together with the detailed structural data, may establish Xyl2 as a platform for future developments of industrially relevant xylanases.Conclusions
F. oxysporum Xyl2 is a GH11 xylanase which is highly active in free form and immobilized onto a variety of solid supports in a wide pH range. Furthermore, immobilization of Xyl2 on certain supports significantly increases its thermal stability. A mechanistic rationale for Xyl2's remarkable catalytic efficiency at alkaline pH is proposed on the basis of two crystallographic structures. Together, these properties render Xyl2 an attractive biocatalyst for the sustainable industrial degradation of xylan.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0605-z) contains supplementary material, which is available to authorized users.
“…When the Nδ2 nitrogen of the imidic acid form of Asn92 extracts a proton from the attacking water, the proton is immediately transferred to the scissile glycosidic bond of substrate through the hydrogen bond network. Meanwhile, in xylanase of GH11, the general acid/base Glu177 receives a proton from the bulk solvent by altering its side‐chain conformation which enables to increase the p K a value of Glu177 . In the present neutron structure analysis of T26H, we found that the Arg145 residue interacting with Glu11 can play an important role in the protonation of the general acid/base in the retaining hydrolysis [Fig.…”
Section: Discussionmentioning
confidence: 79%
“…Based on this observation, we suggest that the proton to be used for the protonation of Glu11 would be supplied by the hydronium ion (Scheme , left). Such proton transfer was also proposed for xylanase, a GH family 11 member . The resulting water molecule can be released to the bulk solvent when the substrate approaches to the active site cleft since it occupies a part of the subsite.…”
T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β‐1,4‐glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09‐Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated Nɛ2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water‐binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high‐resolution X‐ray structure of T26H at 1.04‐Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.
“…To probe the structural changes that occur upon internal aldimine SB protonation, we obtained a low-pH X-ray structure of AAT in the absence of substrate, in which both chains are in the internal aldimine state. Acidifying AAT crystals with acetic acid vapour, using a procedure described previously 23 , 24 , yielded a 1.9 Å room temperature X-ray structure at pH ~4.0 (PDB ID 5VK7; Table 1 ). When the geometries of the internal aldimines (chain B) from the pH 7.5 and pH 4.0 structures were compared, two major differences were evident.…”
Enzymes dependent on pyridoxal 5′-phosphate (PLP, the active form of vitamin B6) perform a myriad of diverse chemical transformations. They promote various reactions by modulating the electronic states of PLP through weak interactions in the active site. Neutron crystallography has the unique ability of visualizing the nuclear positions of hydrogen atoms in macromolecules. Here we present a room-temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase, which was reacted in situ with α-methylaspartate. In one monomer, the PLP remained as an internal aldimine with a deprotonated Schiff base. In the second monomer, the external aldimine formed with the substrate analog. We observe a deuterium equidistant between the Schiff base and the C-terminal carboxylate of the substrate, a position indicative of a low-barrier hydrogen bond. Quantum chemical calculations and a low-pH room-temperature X-ray structure provide insight into the physical phenomena that control the electronic modulation in aspartate aminotransferase.
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