Crystal Structure of β-d-Xylosidase from Thermoanaerobacterium saccharolyticum, a Family 39 Glycoside Hydrolase

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Abstract

1,4-β-d-Xylan is the major component of plant cell-wall hemicelluloses. β-d-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. β-d-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any β-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum β-xylosidase comprises three distinct domains; a catalytic domain of the canonical (β/α)8-barrel fold, a β-sandwich domain, and a small α-helical domain. We have determined the structure in two forms: d-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-α-d-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.

Introduction

1,4-β-d-Xylan is the second most abundant carbohydrate polymer in nature and represents the most abundant polysaccharide in plant cell-wall hemicelluloses. Xylan is composed of a backbone of β-1,4-linked d-xylopyranosyl units harboring arabinofuranose, glucuronic acid, methylglucuronic acid, and acetyl side-groups. Its enzymatic degradation has attracted the attention of the pulp and paper industry because of cost and environmental considerations.1 Complete breakdown of xylan requires the action of many hydrolytic enzymes: (i) α-N-arabinofuranosidase, α-glucuronidase, and acetylesterase cleave the lateral substituents; (ii) endo-1,4-β-xylanase randomly cleaves the β-1,4 bonds in the xylan backbone, producing xylo-oligosaccharides, xylobiose, and xylose; and (iii) β-xylosidase (or 1,4-β-d-xylan xylohydrolase; EC 3.2.1.37) hydrolyzes the resulting xylo-oligosaccharides at their non-reducing end, producing xylose.2

β-Xylosidases from various organisms have been grouped into glycoside hydrolase (GH) families 3, 39, 43, 52, and 54 in the CAZy database†.3 Whereas the hydrolytic reaction by family 43 β-xylosidases results in inversion of the anomeric configuration,4 β-xylosidases of GH families 3, 39, 52 and 54 operate via a retaining mechanism.5 The catalytic mechanism of hen egg-white lysozyme, a retaining β-glycosidase, was shown to involve a covalent glycosyl-enzyme intermediate as postulated by Koshland,6 instead of the oxocarbenium-ion intermediate, which is a key feature of the widely held “Phillips” mechanism.7

β-Xylosidase from the thermophilic anaerobe Thermoanaerobacterium saccharolyticum, a 500 residue protein (monomer Mr=58,606), is one of the best-characterized members of GH family 39. The human lysosomal glycosyl hydrolase α-l-iduronidase, which shows significant sequence similarity to T. saccharolyticum β-xylosidase (108 identical residues for residues 77–482 of β-xylosidase), also belongs to GH family 39, and its deficiency causes the disease mucopolysaccharidosis I.8 The xynB gene encoding T. saccharolyticum β-xylosidase has been expressed at high levels in Escherichia coli and the recombinant enzyme was shown to be optimally active at 343 K.2 T. saccharolyticum β-xylosidase displays a significant transglycosylating activity, a property often found among GHs operating via a retaining mechanism. Its transglycosylation activity shows little regiospecificity, since it can synthesize β-1,2-, β-1,3-, and β-1,4-linked xylo-oligosaccharides.5

The residue acting as the catalytic nucleophile in T. saccharolyticum β-xylosidase was identified as Glu277 by active-site labeling studies,9 while the acid/base catalyst was identified as Glu160 in the highly conserved sequence Asn-Glu-Pro through both labeling and mutagenesis studies.10 A hydrophobic cluster analysis of the protein sequences suggested that family 39 GH members share a similar catalytic domain of the (β/α)8-barrel fold, in agreement with its assignment as a member of clan GH-A of the glycosyl hydrolases.11 However, no detailed structural information is available on any member of GH family 39 or on any β-xylosidase of the five GH families. Crystallization of a fungal β-xylosidase from Trichoderma reesei, a member of GH family 3 and T. saccharolyticum β-xylosidase has been reported.12., 13. To obtain structural information necessary for understanding the catalytic mechanism, we have determined the crystal structure of T. saccharolyticum β-xylosidase in two forms: a d-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-α-d-xylosyl (2F-xylosyl)-xylosidase intermediate. The structure of the covalent reaction intermediate provides direct structural evidence supporting the proposed catalytic mechanism and provides insight into other residues involved in catalysis.9

Section snippets

Structure determination

T. saccharolyticum β-xylosidase crystallized into two different crystal forms under similar crystallization conditions: form A (space group P43212, with two monomers per asymmetric unit) and form B (P212121, with four monomers per asymmetric unit). It crystallized into form A in the presence of d-xylose,13 whereas it crystallized into form B when it was incubated with the inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-xyloside (2F-DNPX).14 The structure of T. saccharolyticum β-xylosidase was

Protein expression, purification and crystallization

The protein was overexpressed in E. coli strain XA-90 harboring the plasmid pXHP3.2., 31. Purification and crystallization of the native protein into form A has been described.13 The SeMet-substituted xylosidase was expressed using the M9 cell culture medium, in which methionine is substituted with SeMet; 10 mM dithiothreitol was added during purification. The SeMet-substituted xylosidase was crystallized under the same conditions as the native protein.13 The SeMet-substituted crystal belongs to

Acknowledgements

We thank Dr Heung Soo Lee and his staff for assistance during data collection at beamline BL-6B of Pohang Light Source, Korea. J.K.Y., H.J.Y., H.J.A. and B.I.L. are supported by the BK21 Fellowship from the Korean Ministry of Education. This work is supported by the Korea Ministry of Science and Technology (NRL-2001, grant number M1-0104-00-0132).

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