The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Carlos Gil-Durán,Inmaculada Vaca,Victoria Guixé,Víctor Castro-Fernández,Fernando D González-Nilo,Romina V Sepúlveda,María-Cristina Ravanal,Maximiliano Rojas,Gloria Levicán,Renato Chávez

doi:10.3390/ijms23042315

Abstract

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-β-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (β/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (β/α)8-barrel domain.

Highlights

Xylan, one of the main components of plant hemicelluloses, is a polysaccharide composed of a backbone of β-D-xylose molecules linked by β-1,4 bonds, which can be substituted by different chemical groups [1]
We generated a homology model for XynA (Figure 1A), and we noticed that in addition to the classical (β/α)8 -barrel fold found in GH10 endoxylanases, XynA contains an intrinsically disordered region (IDR) of 29 amino acids at its amino end (Figure 1B), a region not found in other endoxylanases (Supplementary Figure S1)
Two of these cases correspond to endoxylanases from family GH11 [23,24], while only one corresponds to an endoxylanase from family GH10 [22]

Summary

Introduction

One of the main components of plant hemicelluloses, is a polysaccharide composed of a backbone of β-D-xylose molecules linked by β-1,4 bonds, which can be substituted by different chemical groups [1]. Endoxylanases (endoβ-1,4-xylanases, E.C. 3.2.1.8) are key players, because they act on the backbone of xylan, hydrolyzing internal β-1,4 linkages between xylopyranosyl residues, giving rise to a diverse array of xilooligosaccharides (XOS) that may include xylobiose, xylotriose, xylotetraose, or longer, which may be or not branched by some substituents [1]. Based on amino acid sequence similarities, endoxylanases have been classified into different glycoside hydrolases (GH) families [2]. Endoxylanases from this family have a classical (β/α)8 -barrel fold [3,4]. Concerning activity, GH10 endoxylanases are versatile, acting on different kinds of xylans and XOS

Methods

Results

Conclusion