Crystal Structure of Bacillus sp. GL1 Xanthan Lyase, Which Acts on the Side Chains of Xanthan

Abstract

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 Å resolution with final R-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal ␣-helical and C-terminal ␤-sheet domains, which constitute incomplete ␣ 5 /␣ 5 -barrel and anti-parallel ␤-sheet structures, respectively. A deep cleft is located in the N-terminal ␣-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg 313 and Tyr 315 residues in the loop from the N-terminal domain and the Arg 612 residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

“…The crystal structures of polysaccharide lyases such as those for pectate (13)(14)(15), alginate (16), hyaluronate (17), chondroitin (18,19), and xanthan (20) have been determined, and the structural and functional relationship of these lyases have been studied. No three-dimensional structure has been clarified for any UGL, however.…”

Superchannel of Bacteria: Biological Significance and New Horizons

“…Several amino acid residues are crucial to the binding of PyrMan through the formation of hydrogen bonds and van der Waals contacts. [45][46][47] Trp148 is parallel to the pyranose ring of the mannose moiety of PyrMan, indicating that the residue undergoes a stacked interaction with the sugar ring. The carboxyl group of the pyruvate moiety in PyrMan is bound directly to Arg313, Tyr315, and Arg612 through the formation of four hydrogen bonds.…”

“…Other than these two regions, a region slightly conserved in family PL-7 lyases is found in -strand SA5, proximate to SA3 and SA4. Amino acid residues, including Tyr and His residues in SA3, SA4, and SA5, conserved in family PL-7 alginate lyases, are responsible for the catalytic actions of the enzymes, as in the cases of polysaccharide lyases such as family PL-5 alginate lyase A1-III, 37,38) and family PL-8 hyaluronate, 39) chondroitin, 40) and xanthan [45][46][47] lyases. In fact, His104, Tyr193, and Tyr199 of PA1167 constitute an active cleft.…”

“…Family PL-8 lyases Bacillus species strain GL1 xanthan lyase (81 kDa, exotype) acts on the side chains of xanthan and liberates pyruvated mannose (PyrMan) through a -elimination reaction. 45) Almost all polysaccharide lyases so far analyzed attack the main chains of polysaccharides in an endolytic manner and release oligosaccharides from the polysaccharide. Xanthan lyase is thus characteristic in that it exolytically attacks the side chains of the polysaccharide, and it might provide useful information as to the structural features causing different substrate specificities and modes of action of polysaccharide lyases.…”

“…[45][46][47] A deep cleft formed in the N-terminalhelical domain at the interface between the two domains constitutes an active center (the substrate-binding site), in which Tyr255 is responsible for the catalytic reaction. Some aromatic and positively-charged amino acid residues are present in the active cleft, and they are responsible for the binding and depolymerization of xanthan, an acidic polymer.…”