Structure-function relationships in human salivary α-amylase: role of aromatic residues in a secondary binding site

Abstract

AbstractHuman salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (i) hydrolysis of starch; (ii) binding to hydroxyapatite; and (iii) binding to bacteria (e.g. viridans streptococci). Oral bacteria utilize the starch hydrolyzing activity of HSAmy to derive their nutrients from dietary starch. Localized acid production by bacteria, through the metabolism of maltose generated by HSAmy, can lead to the dissolution of tooth enamel, a critical step in dental caries formation. HSAmy is a component of the acquired enamel pellicle and is used by Streptococcus gordonii to colonize the oral cavity. Although the active site of HSAmy for starch hydrolysis is well characterized, the regions responsible for the bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one of the secondary saccharide-binding sites harboring the aromatic residues W316 and W388, may play an important role in bacterial binding. To test this hypothesis, the aromatic residues W316 and W388 were mutated to alanine. The wild type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch binding and bacterial binding. Our results clearly showed that (i) the mutants W316A and W388A were not impaired in starch binding or bacterial binding; (ii) mutation of aromatic residues at these sites does not alter the overall conformation of the molecule; and (iii) the hydrolytic activity of the enzyme is unaffected against starch as substrates but reduced significantly against oligosaccharides.