Significant enhancement in the binding of p‐nitrophenyl‐β‐d‐xylobioside by the E128H mutant F/10 xylanase from Streptomyces olivaceoviridis E‐86

Abstract

Mutagenesis studies were carried out to examine the effects of replacement of either the nucleophile Glu-236 or the acid/base Glu-128 residue of the F/10 xylanase by a His residue. To our surprise, the affinity for the p-nitrophenyl-L L-D-xylobioside substrate was increased by 10 3 -fold in the case of the mutant E128H enzyme compared with that of the wild-type F/10 xylanase. The catalytic activity of the mutant enzymes was low, despite the fact that the distance between the nucleophilic atom (an oxygen in the native xylanase and a nitrogen in the mutant) and the K K-carbon was barely changed. Thus, the alteration of the acid/base functionality (Glu-128 to His mutation) provided a significantly favorable interaction within the E128H enzyme/substrate complex in the ground state, accompanying a reduction in the stabilization effect in the transition state.z 1999 Federation of European Biochemical Societies.

Crystal Structures of Decorated Xylooligosaccharides Bound to a Family 10 Xylanase from Streptomyces olivaceoviridis E-86

“…A xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A, formally known as FXYN; 45 kDa) has been extensively characterized (3)(4)(5)(6)(7). SoXyn10A consists of a family GH10 catalytic module and a family 13 CBM (referred as SoCBM13, formerly xylan-binding domain), which are joined by a Gly/Pro-rich linker region.…”

Substrate Recognition of a Family 10 Xylanase from Streptomyces olivaceoviridis E-86: A Study by Site-directed Mutagenesis to Make an Hindrance around the Entrance toward the Substrate-binding Cleft

“…The details of the method are as described previously. 13) Gene expression and protein purification. For expression in Escherichia coli and purification of the SoXyn10A and mutant enzymes, the pET expression system (Novagen !…”

“…The circular dichroism (CD) spectra of SoXyn10A and mutant enzymes were acquired using the conditions reported previously. 13,14) Steady state kinetics were investigated as previously reported, 13) briefly the reaction mixture containing 250 µL of the substrate (0.05 5 mM), 150 µL of McIlvaine buffer (a mixture of 0.1 M citric acid and 0.2 M Na2 HPO4, pH 7.0), 50 µL of 1% (w v) bovine serum albumin (BSA) was incubated at 30 C for 5 min and then 50 µL of enzyme solution (0.005 0.1 mg mL) was added. For the hydrolysis of p nitrophenyl β D xyloside (PNP X), reaction mixtures containing 250 µL of the substrate (0.5 mM), 150 µL of McIlvaine buffer, 50 µL of 1% (w v) BSA was incubated at 45 C for 5 min and then 50 µL of enzyme solution (1.0 mg mL) was added.…”

Structure and Function of Carbohydrate-Binding Module Families 13 and 42 of Glycoside Hydrolases, Comprising a β-Trefoil Fold

“…5) CBM13 has been structurally and biochemically analyzed for the first time within intact GHs (Streptomyces endo-1,4--xylanases). 4,[6][7][8] It shows specificity for the backbone of xylan, but it is found not only in xylanases (EC 3.2.1.8), but also in other GHs such as endo-1,3(4)--glucanase (EC 3.2.1.6), 9,10) Biosci. Biotechnol.…”