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Vol 44(2010) N 4 p. 591-595;
I.A. Dvortsov1*, N.A. Lunina1, V.V. Zverlov1,2, G.A. Velikodvorskaya1

Substrate-Binding Properties of the Family 54 Module of Clostridium thermocellum Lic16A Laminarinase

1Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia
2Department of Microbiology, Technische Universität München, Freising, 85350, Germany

*dvortsov@img.ras.ru
Received - 2009-11-17; Accepted - 2010-01-19

Endo-β-1,3-1,4-glucanase Lic16A of the moderate thermophilic anaerobe Clostridium thermocellum has a complex multimodular structure. In addition to the catalytic module, Lic16A contains eight auxiliary modules, five of which are substrate-binding modules. The new family 54 substrate-binding module CBM54 (25.2 kDa), which is at the N terminus of the enzyme, has a cleavage site in its N-terminal part, and its cleavage yields a shortened module CBM54C (17.2 kDa) in vivo and in vitro. CBM54C was cloned in Escherichia coli and purified to electrophoretic homogeneity. The binding constants of CBM54C to xylan, chitin, insoluble yeast cell wall β-glucan, and bacterial crystalline cellulose were of the same order of magnitude as for CBM54. However, CBM54C did not bind pustulan, avicel, and chitosan, in contrast to CBM54. Calcium ions restored the ability of CBM54C to bind these three carbohydrates. CBM54 substrate binding promiscuity suggested multiple binding sites, some of them being Ca2+ dependent. The Ca2+-independent sites for avicel, pustulan and chitosan were localized to the spontaneously split-off N-terminal part (8 kDa) of CBM54. The arrangement of Ca2+-dependent and Ca2+-independent binding sites for various substrates was suggested.

Clostridium thermocellum, laminarinase, multimodular proteins, substrate-binding modules, xylan, chitin, β-glucan, binding sites



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