Chandran Ragunath

Probing the role of aromatic residues at the secondary saccharide binding sites of human salivary α-amylase in substrate hydrolysis and bacterial binding

Human salivary alpha-amylase (HSAmy) has three distinct functions relevant to oral health: (1) hydrolysis of starch, (2) binding to hydroxyapatite (HA), and (3) binding to bacteria (e.g., viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well-characterized, the regions responsible for 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 or more of the secondary saccharide-binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A, and W284A), double (Y276A/W284A and W316A/W388A), or multiple [W134A/W203A/Y276A/W284A/W316A/W388A; human salivary alpha-amylase aromatic residue multiple mutant (HSAmy-ar)] mutations. The crystal structure of HSAmy-ar as an acarbose complex was determined at a resolution of 1.5 A and compared with the existing wild-type acarbose complex. The wild-type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch-binding activity, HA-binding activity, and bacterial binding activity. Our results clearly showed that (1) mutation of aromatic residues does not alter the overall conformation of the molecule; (2) single or double mutants showed either moderate or minimal changes in both starch-binding activity and bacterial binding activity, whereas HSAmy-ar showed significant reduction in these activities; (3) starch-hydrolytic activity was reduced by 10-fold in HSAmy-ar; (4) oligosaccharide-hydrolytic activity was reduced in all mutants, but the action pattern was similar to that of the wild-type enzyme; and (5) HA binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide-binding sites in HSAmy play a critical role in bacterial binding and in starch-hydrolytic functions of HSAmy.

Role of active-site residues of dispersin B, a biofilm-releasing β-hexosaminidase from a periodontal pathogen, in substrate hydrolysis

Dispersin B (DspB), a family 20 β‐hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves β(1,6)‐linked N‐acetylglucosamine polymer. In order to understand the substrate specificity of DspB, we have undertaken to characterize several conserved and nonconserved residues in the vicinity of the active site. The active sites of DspB and other family 20 hexosaminidases possess three highly conserved acidic residues, several aromatic residues and an arginine at subsite −1. These residues were mutated using site‐directed mutagenesis and characterized for their enzyme activity. Our results show that a highly conserved acid pair in β‐hexosaminidases D183 and E184, and E332 play a critical role in the hydrolysis of the substrates. pH activity profile analysis showed a shift to a higher pH (6.8) in the optimal activity for the E184Q mutant, suggesting that this residue might act as the acid/base catalyst. The reduction in kcat observed for Y187A and Y278A mutants suggests that the Y187 residue (unique to DspB) located on a loop might play a role in substrate specificity and be a part of subsite +1, whereas the hydrogen‐bond interaction between Y278A and the N‐acetyl group might help to stabilize the transition state. Mutation of W237 and W330 residues abolished hydrolytic activity completely suggesting that alteration at these positions might collapse the binding pocket for the N‐acetyl group. Mutation of the conserved R27 residue (to R27A or R27K) also caused significant reduction in kcat suggesting that R27 might be involved in stabilization of the transition state. From these results, we conclude that in DspB, and possibly in other structurally similar family 20 hydrolases, some residues at the active site assist in orienting the N‐acetyl group to participate in the substrate‐assisted mechanism, whereas other residues such as R27 and E332 assist in holding the terminal N‐acetylglucosamine during the hydrolysis.

Subsite mapping of human salivary α-amylase and the mutant Y151M

This study characterizes the substrate‐binding sites of human salivary α‐amylase (HSA) and its Y151M mutant. It describes the first subsite maps, namely, the number of subsites, the position of cleavage sites and apparent subsite energies. The product pattern and cleavage frequencies were determined by high‐performance liquid chromatography, utilizing a homologous series of chromophore‐substituted maltooligosaccharides of degree of polymerization 3–10 as model substrates. The binding region of HSA is composed of four glycone and three aglycone‐binding sites, while that of Tyr151Met is composed of four glycone and two aglycone‐binding sites. The subsite maps show that Y151M has strikingly decreased binding energy at subsite (+2), where the mutation has occurred (−2.6 kJ/mol), compared to the binding energy at subsite (+2) of HSA (−12.0 kJ/mol).

Evidence for pentagalloyl glucose binding to human salivary α-amylase through aromatic amino acid residues

We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K(EI)=2.6 microM and K(ESI)=3.9 microM were determined from Lineweaver-Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.

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

Human 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.