Structure of Human Salivary α-Amylase at 1.6 Å Resolution: Implications for its Role in the Oral Cavity

Abstract

Salivary a-amylase, a major component of human saliva, plays a role in the initial digestion of starch and may be involved in the colonization of bacteria involved in early dental plaque formation. The three-dimensional atomic structure of salivary amylase has been determined to understand the structure-function relationships of this enzyme. This structure was refined to an R value of 18.4% with 496 amino-acid residues, one calcium ion, one chloride ion and 170 water molecules. Salivary amylase folds into a multidomain structure consisting of three domains, A, B and C. Domain A has a (/3/a)8-barrel structure, domain B has no definite topology and domain C has a Greek-key barrel structure. The Ca 2+ ion is bound to Asnl00, Arg158, Asp167, His201 and three water molecules. The C1-ion is bound to Arg195, Asn298 and Arg337 and one water molecule. The highly mobile glycine-rich loop 304-310 may act as a gateway for substrate binding and be involved in a 'trap-release' mechanism in the hydrolysis of substrates. Strategic placement of calcium and chloride ions, as well as histidine and tryptophan residues may play a role in differentiating between the glycone and aglycone ends of the polysaccharide substrates. Salivary amylase also possesses a suitable site for binding to enamel surfaces and provides potential sites for the binding of bacterial adhesins.

Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

“…The wide range of known three-dimensional structures of a-amylases from Aspergillus oryzae (Matsuura et al, 1984;Swift et al, 1991), Aspergillus niger (Boel et al, 1990;Brady et al, 1991), porcine pancreas (Buisson et al, 1987;Qian et al, 1993;Larson et al. 1994), barley seeds (Kadziola et al, 1994), Bacillus lichenifomis (Machius et al, 1995), human pancreas (Brayer et al, 1995), and human salivary (Ramasubbu et al, 1996) constitute an ideal system for studies of adaptation of enzymes to extreme conditions on a molecular level. The three-dimensional structures obtained and described in this paper provide new insights into the mechanism of cold adaptation.…”

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

“…These Cl − -dependent α-amylases are usually specific to all mammalian species, but are also found in a few types of bacteria, such as Pseudoalteromonas haloplanktis (D' Amico et al 2000). The solved structures of chloride-dependent α-amylases from human salivary (Ramasubbu et al 1996) and pancreatic (Brayer et al 1995) glands, porcine pancreas (Qian et al 1993;Larson et al 1994), Tenebrio molitor (Strobl et al 1998), and P. haloplanktis (Aghajari et al 2002) contain the ion at a common site, which is located close to the center of the (β/α) 8 -barrel and in the near vicinity of the catalytic site. The protein ligands for the chloride ion and the interaction network within the active site have been elucidated (Aghajari et al 2002).…”

“…A study of P. haloplanctis α-amylase has revealed that Cl − can interact with active site carboxylates and the protective effect against Ca 2+ inhibition is consistent with the appearance of a protonated carboxyl group upon Cl − binding (Feller et al 1996). The chloride-binding site of mammalian pancreatic and salivary α-amylases consists of Arg195, Asn298, the side-chain amines of Arg337, and a water molecule (Brady et al 1991;Qian et al 1993;Lawson et al 1994;Ramasubbu et al 1996). This binding site of Bl TreA differs from them by a tyrosine residue instead of Arg337 (Fig.…”

“…The hydrogen bonds are indicated by dashed lines. Ramasubbu et al 1996). These protein ligands coordinated to chloride ion are part of highly conserved regions of Cl − -dependent group of the family GH13 enzymes (Fig.…”

“…Three-dimensional structures of several GH13 family enzymes, including α-amylases (Matsuura et al 1984;Brady et al 1991;Qian et al 1993;Kadziola et al 1994;Brayer et al 1995;Machius et al 1995;Ramasubbu et al 1996), cyclodextrin glucanotransferases (Klein & Schulz 1991;Kubota et al 1991;Lawson et al 1994;Knegtel et al 1996), and oligo-1,6-glucosidase (Kizaki et al 1993;Watanabe et al 1997) are already available. Collectively, family GH13 enzymes have certain criteria of evolution conservatory: (1) a very low degree of sequence similarities, located at or near strands β2, β3, β4, β5, and β7, and at a short region close to the C-terminus of domain B, serving as fingerprints (Kizaki et al 1993;Knegtel et al 1996); (2) an insertion of domain B between strand β3 and helix α3 of the catalytic (β/α) 8 -barrel (MacGregor 1993;Janeček 1994;Svensson 1994); and (3) three fully conserved catalytic residues Asp206, Glu230, and Asp297 (numbering as in Taka-amylase A) Strokopytov et al 1995).…”