Structural and functional definition of the human chitinase chitin-bindingdomain

Mammalian chitinase, a chitinolytic enzyme expressed by macrophages, has be en detected in atherosclerotic plaques and is elevated in blood and tissues of guinea pigs infected with Aspergillus, Its normal physiological functio n is unknown, To understand how the enzyme interacts with its substrate, we have characterized the chitin-binding domain, The C-terminal 49 amino acid s make up the minimal sequence required for chitin binding activity, The ab sence of this domain does not affect the ability of the enzyme to hydrolyze the soluble substrate, triacetylchitotriose, but abolishes hydrolysis of i nsoluble chitin, Within the minimal chitin-binding domain are six cysteines ; mutation of any one of these to serine results in complete loss of chitin binding activity. Analysis of purified recombinant chitin-binding domain r evealed the presence of three disulfide linkages. The recombinant domain bi nds specifically to chitin but does not bind chitosan, cellulose, xylan, be ta-1,3-glucan, beta-1,3-1,4-glucan, or mannan, Fluorescently tagged chitin- binding domain was used to demonstrate chitin-specific binding to Saccharom yces cerevisiae, Candida albicans, Mucor rouxii, and Neurospora crassa, The se experiments define structural features of the minimal domain of human ch itinase required for both specifically binding to and hydrolyzing insoluble chitin and demonstrate relevant binding within the context of the fungal c ell wall.