Conference Paper: Structure-function studies of glycosyl hydrolases[Show abstract] [Hide abstract] ABSTRACT: [MS5-P06] Structure-function Studies of Glycosyl Hydrolases. Jan Dohnálek,a Jarmila Dušková,a Galina Tiščenko,a Tereza Skálová,a Petr Kolenko, a Andrea Štěpánková,b Tomáš Podzimek,c Petra Lipovová,c Jindřich Hašek,a Jiří Šimůnekd a Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovského nám. 2, 162 06 Prague 6, Czech Republic, b Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University, Břehová 7, 115 19 Praha 1, Czech Republic, c Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Czech Republic, d Institute of Animal Physiology and Genetics AS CR, Vídeňská 1083, 142 00 Prague 4, Czech Republic. E-mail: firstname.lastname@example.org Glycosyl hydrolases have been in the centre of our structure-function studies for many years because of their high potential for biotechnological and biomedical applications. Our original structural study of a GH2 β-galactosidase from psychrophilic bacterium Arthrobacter sp. C2-2 revealed its unique hexameric arrangement  and more data have now been gathered on ligand binding in its active site and accompanying structural changes of the enzyme. This cold active β-galactosidase capable of maintaining significant acitivity at 10° C can bind its saccharide ligands in a shallow binding mode even if the key residue for this binding mode, Trp999 of mesophilic E. coli β-galactosidase is in the cold-active enzyme replaced by Cys999. X-ray structures of three complexes of the enzyme with a product, transition state analogue, and inhibitor identify structural changes near and apart from the active site connected with ligand binding . Chitinases of GH18 from a human commensal bacterium Clostridium paraputrificum J4 can be potentially exploited in treatment of diseases caused by pathogenic fungi. This bacterium discovered, isolated and studied by our team attracted our attention by a large number of chitinolytic enzymes secreted into medium . Recently, we have sequenced the genome of the bacterium and discovered at least 12 protein coding sequences annotated as chitinolytic enzymes. Chitinase Chit62J4 of GH18 is a four-domain enzyme, with mostly exochitinase activity and its catalytic domain highly similar to Nctu2 from Bacillus cereus. ChitBJ4 is homologous to ChiB of Clostridium paraputrificum M-21. Several of the enzymes of the chitinolytic complex undergo crystallization experiments and tests of their effects on pathogens [4,5]. As the catalytic mechanisms have been explained our work is focused on the role of the additional domains on activity and on the catalytic efficiency of the whole enzymatic complex in chitin degradation. Support by the Czech Science Foundation (no. 310/09/1407) and by the Czech Ministry of Education, Youth and Sports (no. EE2.3.30.0029) is acknowledged.  Skálová, T., Dohnálek, J., Spiwok, V., Lipovová, P., Vondráčková, E., Petroková, H., Dušková, J., Strnad, H., Králová, B., Hašek, J. (2005) J. Mol. Biol. 353, 282-294.  Štěpánková, A. (2012) X-ray structural analysis of enzymes from extremophiles and their complexes with bound ligands. PhD thesis, Czech Technical University in Prague.  Šimůnek J., Kopečný J., Hodrová B., Bartoňová H. (2002) Folia Microbiol. 47, 559- 564.  Dušková J., Tishchenko G., Ponomareva E., Šimůnek J., Koppová I., Skálová T., Štěpánková A., Hašek J., Dohnálek J. (2011) Acta Biochim. Pol. 58, 261-263.  Šimůnek J., Koppová I., Tischchenko G., Dohnálek J., Dušková J. (2012) Folia Microbiol. 57, 335-339. Keywords: chitinase, beta-galactosidase, structure-function relationship
- [Show abstract] [Hide abstract] ABSTRACT: A strictly anaerobic mesophilic chitinolytic bacterial strain identified as Clostridium paraputrificum J4 was isolated from human feces. In response to various types of growth substrates, the bacterium produced an array of chitinolytic enzymes representing significant components of the J4 strain secretome. The excreted active proteins were characterized by estimating the enzymatic activities of endochitinase, exochitinase, and N-acetylglucosaminidase induced by cultivation in medium M-10 with colloidal chitin. The enzyme activities produced by J4 strain cultivated in medium M-10 with glucose were significantly lower. The spectrum of extracellularly excreted proteins was separated by SDS-PAGE. The chitinase variability was confirmed on zymograms of renatured SDS-PAGE. The enzymes were visualized under ultraviolet light by using 4-methylumbelliferyl derivatives of N-acetyl-β-D: -glucosaminide, N,N´-diacetyl-β-D: -chitobiose, or N,N´,N˝-triacetyl-β-D: -chitotriose for β-N-acetylglucosaminidase, chitobiosidase, or endochitinase activities, respectively. Protein components of the secretome were separated by 2D-PAGE analysis. The distinct protein bands were excised, isolated, and subsequently characterized by using MALDI-TOF/TOF tandem mass spectrometry. The final identification was performed according to sequence homology by database searching.