STRUCTURAL BASIS FOR SUBSTRATE RECOGNITION BY GH30 GLUCURONOXYLANASE FROM ERWINIA CHRYSANTHEMI

Ľubica Urbániková¹, Mária Vršanská², Kristian Bertel Rømer Mørkeberg Krogh³, Tine Hoff³ and Peter Biely²

¹Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia

²Institute of Chemistry, Center of Glycomics, Slovak Academy of Sciences, Bratislava, Slovakia

³Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark

The important industrial enzymes, endo-b-1,4-xylanases (EC 3.2.1.8), have been classified into several glycoside hydrolase (GH) families based on hydrophobic cluster analysis, three dimensional structure and mode of action [1]. Best known enzymes belong to GH families 10 and 11. These enzymes do not seem to be specialized for hydrolysis of a particular xylan. Unique xylanases were assigned to GH family 30 [2]. Some of bacterial GH30 xylanases are specialized for hydrolysis of xylans containing D-glucuronosyl or 4-O-methyl D-glucuronosyl side residues. With enzyme species from Bacillus subtilis (Xyn C) and Erwinia chrysanthemi (XynA) it was clearly demonstrated that the cleavage of xylan main chain is dependent on the presence of uronic acid side residues [3-7].

Recently, the structure of xylanase A from the phytopathogenic bacterium E. chrysanthemi with free active site was reported [7]. Here we report the crystal structure of xylanase A in the complex with aldotetraouronic acid MeGlcA²Xyl_3, an analogue of the product of enzymatic reaction. The crystal structure of the enzyme-ligand complex was solved at 1.39 Å resolution [8]. The ligand xylotriosyl moiety occupies three earlier recognized subsites -1, -2 and -3, while the MeGlcA residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the negatively charged uronic acid carboxylate with positively charged guanidinium group of Arg293. The interaction of the enzyme with MeGlcA residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear b-1,4-xylooligosaccharides which are hydrolyzed by the enzyme at a negligible rate. In the close proximity to the catalytic amino-acids Glu165 and Glu253 an electron density has been found into which a molecule of imidazole was modeled. Imidazole is a part of the crystallization buffer. It is suggested that imidazole occupies +1 subsite binding xylose or xylosyl residues of the enzyme-cleaved substrates. The structure of the protein-MeGlcA²Xyl₃ complex was used for calculation of the binding energy for the ligand itself and for its analogues.

Structural analysis, energy calculations and experimentally measured specific activity of the enzyme on various substrates are used to answer the question why the enzyme does not attack efficiently linear b-1,4-linked xylooligosaccharides.

This work was supported by VEGA grants 2/0001/10 and 2/0165/08 from the Slovak Academy of Sciences. We acknowledge the EMBL X13 beamline at the DORIS storage ring, DESY, Hamburg for providing us with synchrotron source facilities.

1 P.M. Coutinho & B.Henrissat, Carbohydrate-active enzymes server at http://afmb.cnrs-mrs.fr/CAZY/index.html.

2 F.J. St. John, J.M. Gonzalez & E. Pozharski, FEBS Letters, 584 (2010) 4435-4441.

3 K. Nishitani & D.J. Nevins, J. Bio.l Chem., 266 (1991) 6539-6543.

4 J.C. Hurlbert & J.F. Preston, J. Bacteriol., 183(2001) 2093-2100.

5 F.J. St John, J.D. Rice & J.F. Preston, J. Bacteriol., 188 (2006) 8617-8626.

6 M. Vršanská, K. Kolenová, V. Puchart & P. Biely, Eur. J. Biochem., 274 (2007) 1666-1677.

7 S.B. Larson, J. Day, A.P. Barba de la Rosa, N.T. Keen & A. McPherson, Biochemistry, 42 (2003) 8411-8422.

8 Ľ. Urbániková, M. Vršanská, K.B.R.M. Krogh, T. Hoff & P. Biely, FEBS J., 278 (2011) submitted