The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships

Sapag, Amalia; Wouters, Johan; Lambert, Christophe; Ioannes, Pablo De; Eyzaguirre, Jaime; Depiereux, Eric

doi:10.1016/s0168-1656(02)00002-0

Cited by 135 publications

(88 citation statements)

References 102 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Thus, the apparent pK a value of the general acid catalyst measured previously by NMR can be considered as an average of the intrinsic pK a s of the two glutamate conformations. Sequence analysis of GH11 enzymes reveals that residues are highly conserved in the pocket surrounding the downward conformation of Glu177 (42). In acidophilic xylanases active well below pH 5.0, the Asn71, Gly178, and Tyr179 residues from this pocket are frequently replaced by Ser, Ala, and Trp, respectively, whereas Asn44 is universally substituted with Asp.…”

Section: Discussionmentioning

confidence: 99%

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan

Parks

Hanson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pK a values of the catalytic Glu residues in both the apo-and substrate-bound states of the enzyme. The calculated pK a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.glycoside hydrolase | protonation state | macromolecular neutron crystallography | xylanase | molecular simulations

show abstract

Section: Discussionmentioning

confidence: 99%

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan

Parks

Hanson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Alignment of the A. niger xylanase with other family 11 xylanases from fungal and bacterial origin showed that the Asn 117 residue was conserved in most sequences (8), indicating that the environment of this amino acid in the three-dimensional structure is more likely to be responsible for the difference in binding. The A. niger xylanase three-dimensional structure comprises a single domain containing one ␣-helix and 13 ␤-strands, which are arranged in two mostly antiparallel ␤-sheets, A and B (Fig.…”

mentioning

confidence: 99%

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Tahir

Berrin

Flatman

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The importance of aromatic and charged residues at the surface of the active site of a family 11 xylanase from Aspergillus niger was evaluated using site-directed mutagenesis. Ten mutant proteins were heterologously produced in Pichia pastoris, and their biochemical properties and kinetic parameters were determined. The specific activity of the Y6A, Y10A, Y89A, Y164A, and W172A mutant enzymes was drastically reduced. The low specific activities of Y6A and Y89A were entirely accounted for by a change in k cat and K m , respectively, whereas the lower values of Y10A, Y164A, and W172A were due to a combination of increased K m and decreased k cat . Tyr 6 , Tyr 10 , Tyr 89 , Tyr 164 , and Trp 172 are proposed as substrate-binding residues, a finding consistent with structural sequence alignments of family 11 xylanases and with the three-dimensional structure of the A. niger xylanase in complex with the modeled xylobiose. All other variants, D113A, D113N, N117A, E118A, and E118Q, retained full wild-type activity. Only N117A lost its sensitivity to xylanase inhibitor protein I (XIP-I), a protein inhibitor isolated from wheat, and this mutation did not affect the fold of the xylanase as revealed by circular dichroism. The N117A variant showed kinetics, pH stability, hydrolysis products pattern, substrate specificity, and structural properties identical to that of the wild-type xylanase. The loss of inhibition, as measured in activity assays, was due to abolition of the interaction between XIP-I and the mutant enzyme, as demonstrated by surface plasmon resonance and electrophoretic titration. A close inspection of the threedimensional structure of A. niger xylanase suggests that the binding site of XIP-I is located at the conserved "thumb" hairpin loop of family 11 xylanases.

show abstract

“…It is noted FF-FR, encoding for 30 amino acids (AA) (GNAYLAVYGWTTSPLIEYYIMETKNLRVTI) and showing a consensus EYYI which matched the conserved region of all xylanases proteins (19). The DNA sequences alignments have a high degree of similarity in nucleic and amino acids with other fungal xylanases (data not shown).…”

Section: Classification Of Xylanase Genementioning

confidence: 93%

Cloning and molecular characterization of a new fungal xylanase gene from Sclerotinia sclerotiorum S2

Ellouze

Loukil²,

Marzouki³

2011

BMB Reports

View full text Add to dashboard Cite

The endoxylanases from family 11: computer analysis of protein sequences reveals important structural and phylogenetic relationships

Cited by 135 publications

References 102 publications

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Specific Characterization of Substrate and Inhibitor Binding Sites of a Glycosyl Hydrolase Family 11 Xylanase fromAspergillus niger

Cloning and molecular characterization of a new fungal xylanase gene from Sclerotinia sclerotiorum S2

Contact Info

Product

Resources

About