STRUCTURE OF RECOMBINANT SALIVARY [[alpha]]-AMYLASE: POSSIBLE ROLE OF THE INDIVIDUAL DOMAINS IN DENTAL PLAQUE FORMATION. Kalaiyarasi Ramalingam, Ching-Chung Tseng, Narayanan Ramasubbu, Michael J. Levine, Department of Oral Biology and Dental Research Institute, School of Dental Medicine, State University of New York at Buffalo, Buffalo, NY 14214, USA

Salivary [[alpha]]-amylase (sAmy) is a multifunctional enzyme involved in the initial hydrolysis of starch and in the colonization of bacteria which leads to dental plaque formation. Amylases are monomeric, calcium binding proteins with a single polypeptide chain folded into three domains and contain the ([[beta]]/[[alpha]])₈-barrel topology. Domains A and B appear to be involved in enzymatic activity. The role of the C domain in either of the functions is unknown. To better understand the functional roles of the various domains of this enzyme we have optimized a baculovirus expression system for the production of recombinant amylase (rAmy) with biochemical and biological properties similar to native sAmy. Crystallization of rAmy was carried out by vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant in the presence of CaCl₂ at pH 9.0. The crystals are of the space group P2₁2₁2₁ with unit cell dimensions of a=52.63, b=75.20 and c=137.11 Å with one molecule per asymmetric unit. A total of 21,505 unique reflections were collected on a R-AXIS II imaging plate system to a resolution of 2.0 Å using two crystals (Rmerge = 9.38%). The structure of rAmy was solved by molecular replacement techniques using the sAmy structure as a starting model. The structure has been refined at 2.5 Å resolution to an R factor of 20% with excellent stereochemistry. The overall topological fold of the molecule is identical to the native enzyme. The comparison of the recombinant amylase structure to other functionally related enzymes has revealed potential sites for bacterial binding. Consequently, we have initiated the generation of mutants targeted against the selective control of bacterial binding and enamel binding.

Work supported by USPHS grants DE10621 and DE08240