ISSN 0026-8933, Molecular Biology, 2010, Vol. 44, No. 4, pp. 591–595. © Pleiades Publishing, Inc., 2010. Original Russian Text © I.A. Dvortsov, N.A. Lunina, V.V. Zverlov, G.A. Velikodvorskaya, 2010, published in Molekulyarnaya Biologiya, 2010, Vol. 44, No. 4, pp. 671–667. 591 INTRODUCTION Clostridium thermocellum has long attracted attention as a source of thermostable enzymes of cellulose degrada- tion. The bacterium utilizes mostly cellulose, cellodex- trins, and β-1,3(1,6; 1,4)-glucans with the use of at least two enzymatic systems, one belonging to the cellulosome and the other belonging to the accessory system consisting of free enzymes [1–7]. The latter includes laminarinase Lic16A. This protein (1323 amino acid residues, 148 kDa) consists of nine modules (Fig. 1). Its properties have been investigated [8]. The enzyme is most active on soluble sub- strates (lichenan and laminarin), least active on insoluble glucans with β-1,3 bonds (pachyman and curdlan), and inactive on β-1,3-1,4-glucan of the yeast cell wall and β- 1,4-glucans (xylan and chitin). Lic16A is moderately ther- mostable, its optimal temperature is 60–70°С. The enzyme occurs in the surface layer of the C. thermocellum cell wall. Carbohydrate-binding module 54 (CBM54) of Lic16A has been studied recently [9]. CBM54 is in region 191–426 of the Lic16A amino acid sequence and is separated from the adjacent modules with PTS boxes, which are flexible linkers. Based on the melting data, CBM54 is a compact protein. The enzyme is capable of binding several polysaccharides. The high- est affinity has been observed for xylan, β-1,3-1,4-glu- can of the yeast cell wall, and, in the presence of Ca 2+ , for chitin. A binding of soluble substrates (lichenan and laminarin) has not been detected. CBM54 is not highly specific for a certain type of substrates; its sub- strates include carbohydrates with β-1,3, β-1,4, and β- 1,6 bonds. The interaction with certain substrates depends on Ca 2+ . A conserved cleavage site has been found in the CBM54 polypeptide chain (Fig. 1). When Lic16A is isolated from the C. thermocellum cell wall, an enzyme of 125, rather than 148, kDa is obtained. Denaturing PAGE reveals two bands corresponding to 120- and 29-kDa proteins during purification of the Lic16A module. The Lic16A cleavage site is in the CBM54 region according to sequencing of the N-terminal region of the protein (120 kDa). Likewise, the isolated CBM54 module partly degrades, cleavage occurring in the same site as in Lic16A [8, 9]. Since CBM54 interacts with a broad range of sub- strates and is relatively large (235 amino acid residues), it is possible to assume that several substrate-binding mod- ules or substrate-binding sites occur in the CBM54 pro- tein sequence. In this work, we studied the distribution of substrate-binding sites along CBM54. Substrate-Binding Properties of the Family 54 Module of Clostridium thermocellum Lic16A Laminarinase I. A. Dvortsov a , N. A. Lunina b , V. V. Zverlov a, b , and G. A. Velikodvorskaya a a Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182 Russia; e-mail: dvortsov@imb.ras.ru b Department of Microbiology, Technische Universitát München, Freising, 85350 Germany Received November 17, 2009 Accepted for publication January 19, 2010 Abstract—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 electro- phoretic 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 Ca 2+ dependent. The Ca 2+ -independent sites for avicel, pustulan and chitosan were localized to the spontaneously split-off N-terminal part (8 kDa) of CBM54. The arrangement of Ca 2+ -dependent and Ca 2+ -independent binding sites for various substrates was suggested. DOI: 10.1134/S002689331004014X Key words: Clostridium thermocellum, laminarinase, multimodular proteins, substrate-binding modules, xylan, chitin, β-glucan, binding sites CELL MOLECULAR BIOLOGY UDC