Carbohydrate-binding properties of a separately folding protein module from beta-1,3-glucanase Lic16A of Clostridium thermocellum.
The multi-modular non-cellulosomal endo-1,3(4)-beta-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca2+. The highest affinity to CBMX was shown for xylan (binding constant K=3.1x10(4) M(-1)), yeast cell wall glucan (K=1.4x10(5) M(-1)) and chitin (K=3.3.10(5) M(-1) in the presence of Ca2+). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.
Dvortsov
Igor A
IA
Institute of Molecular Genetics RAS, 2 Kurchatov Square, 123182 Moscow, Russian Federation.
Lunina
Natalia A
NA
Institute of Molecular Genetics RAS, 2 Kurchatov Square, 123182 Moscow, Russian Federation.
Chekanovskaya
Ludmila A
LA
Institute of Molecular Genetics RAS, 2 Kurchatov Square, 123182 Moscow, Russian Federation.
Schwarz
Wolfgang H
WH
Department of Microbiology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany.
Zverlov
Vladimir V
VV
Department of Microbiology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany.|Institute of Molecular Genetics RAS, 2 Kurchatov Square, 123182 Moscow, Russian Federation.
Velikodvorskaya
Galina A
GA
Institute of Molecular Genetics RAS, 2 Kurchatov Square, 123182 Moscow, Russian Federation.
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry / Q000378:metabolism; D005936:Glucans / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
D005936:Glucans; D011134:Polysaccharides; D014990:Xylans; D002686:Chitin; D043326:Glucan 1,3-beta-Glucosidase; D002118:Calcium