Evidence for pentagalloyl glucose binding to human salivary alpha-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.

Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-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.

Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans.

Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrix-encased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is poly-beta-1,6-N-acetyl-d-glucosamine (PGA), a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report, we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to A. actinomycetemcomitans biofilm cells and could be efficiently separated from the cells only by phenol extraction. A. actinomycetemcomitans PGA copurified with LPS on a gel filtration column. (1)H NMR spectra of purified A. actinomycetemcomitans PGA were consistent with a structure containing a linear chain of N-acetyl-d-glucosamine residues in beta(1,6) linkage. Genetic analyses indicated that all four genes of the pgaABCD locus were required for PGA production in A. actinomycetemcomitans. PGA mutant strains still formed biofilms in vitro. Unlike wild-type biofilms, however, PGA mutant biofilms were sensitive to detachment by DNase I and proteinase K. Treatment of A. actinomycetemcomitans biofilms with the PGA-hydrolyzing enzyme dispersin B made them 3 log units more sensitive to killing by the cationic detergent cetylpyridinium chloride. Our findings suggest that PGA, extracellular DNA and proteinaceous adhesins all contribute to the structural integrity of the A. actinomycetemcomitans biofilm matrix.

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

Dispersin B (DspB), a family 20 beta-hexosaminidase from the oral pathogen Aggregatibacter actinomycetemcomitans, cleaves beta(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 beta-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 k(cat) 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 k(cat) 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.

Induction of cathelicidin in normal and CF bronchial epithelial cells by 1,25-dihydroxyvitamin D(3).

BACKGROUND: Antimicrobial peptides (AMPs) such as cathelicidins contribute to initial defense of the airway against inhaled pathogens. Recent studies have shown that the hormonally active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) up-regulates AMP gene expression in several established cell lines. Furthermore, serum levels of vitamin D are often deficient in cystic fibrosis (CF) patients. METHODS: We investigated the effect of 1,25(OH)(2)D(3) on AMP mRNA levels in primary cultures of normal human bronchial epithelial (NHBE) cells by real-time PCR, and protein levels by Western blot. Antimicrobial activity of airway surface fluid from these cells was measured by in vitro assay against laboratory strains of bacteria. RESULTS: Treatment of NHBE cells with 1,25(OH)(2)D(3) (10(-8)M), resulted in a 10-fold up-regulation of cathelicidin mRNA levels after 12 h, which was augmented 2-fold with co-incubation of 1 mM Calcium. Moreover, 1,25(OH)(2)D(3) induced antimicrobial activity against the airway pathogens Bordetella bronchiseptica and Pseudomonas aeruginosa. 1,25(OH)(2)D(3) induced cathelicidin mRNA expression equally in both normal and CF bronchial epithelial cells. CONCLUSIONS: Elucidation of the effect of 1,25(OH)(2)D(3) on cathelicidin expression in NHBE cells and CF bronchial epithelial cells will aid in the development of novel therapeutic agents for treatment of airway infections in CF.

Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae.

Most field isolates of the swine pathogen Actinobacillus pleuropneumoniae form tenacious biofilms on abiotic surfaces in vitro. We purified matrix polysaccharides from biofilms produced by A. pleuropneumoniae field isolates IA1 and IA5 (serotypes 1 and 5, respectively), and determined their chemical structures by using NMR spectroscopy. Both strains produced matrix polysaccharides consisting of linear chains of N-acetyl-D-glucosamine (GlcNAc) residues in beta(1,6) linkage (poly-beta-1,6-GlcNAc or PGA). A small percentage of the GlcNAc residues in each polysaccharide were N-deacetylated. These structures were nearly identical to those of biofilm matrix polysaccharides produced by Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. PCR analyses indicated that a gene encoding the PGA-specific glycoside transferase enzyme PgaC was present on the chromosome of 15 out of 15 A. pleuropneumoniae reference strains (serotypes 1-12) and 76 out of 77 A. pleuropneumoniae field isolates (serotypes 1, 5 and 7). A pgaC mutant of strain IA5 failed to form biofilms in vitro, as did wild-type strains IA1 and IA5 when grown in broth supplemented with the PGA-hydrolyzing enzyme dispersin B. Treatment of IA5 biofilms with dispersin B rendered them more sensitive to killing by ampicillin. Our findings suggest that PGA functions as a major biofilm adhesin in A. pleuropneumoniae. Biofilm formation may have relevance to the colonization and pathogenesis of A. pleuropneumoniae in pigs.

Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from prosthetic hip and knee joint infections.

Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. As the use of biofilm-associated factors as vaccines is critically restricted by their prevalence in natural staphylococcal populations we studied the distribution of genes involved in biofilm formation, the biofilm phenotype and production of polysaccharide intercellular adhesin (PIA) in clonally independent Staphylococcus aureus and Staphylococcus epidermidis strains isolated from prosthetic joint infections after total hip or total knee arthroplasty. Biofilm formation was detected in all S. aureus and 69.2% of S. epidermidis strains. Importantly, 27% of biofilm-positive S. epidermidis produced PIA-independent biofilms, in part mediated by the accumulation associated protein (Aap). Protein-dependent biofilms were exclusively found in S. epidermidis strains from total hip arthroplasty (THA). In S. aureus PIA and proteins act cooperatively in biofilm formation regardless of the infection site. PIA and protein factors like Aap are of differential importance for the pathogenesis of S. epidermidis in prosthetic joint infections (PJI) after THA and total knee arthroplasty (TKA), implicating that icaADBC cannot serve as a general virulence marker in this species. In S. aureus biofilm formation proteins are of overall importance and future work should focus on the identification of functionally active molecules.

Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms.

Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.

Enzymatic detachment of Staphylococcus epidermidis biofilms.

The gram-positive bacterium Staphylococcus epidermidis is the most common cause of infections associated with catheters and other indwelling medical devices. S. epidermidis produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces. We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans rapidly and efficiently removed S. epidermidis biofilms from plastic surfaces. The enzyme worked by releasing extracellular slime from S. epidermidis cells. Precoating surfaces with the enzyme prevented S. epidermidis biofilm formation. Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents.

Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.

Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity.

In the mechanism of hydrolysis of starch by alpha-amylases, a conserved water molecule bridging two catalytic residues has been implicated. In human salivary alpha-amylase (HSAmy), this water (W641), observed in many alpha-amylase structures, is part of a chain of water molecules. To test the hypothesis that W641 may be involved in the mechanism, Phe256 in the close vicinity was mutated to a Trp residue. X-ray structure of F256W complexed to 2-amino-2-(hydroxyethyl)-1,3-propanediol at 2.1A revealed that the water chain is disrupted. In the F256W structure exhibits a positional shift in His305, characteristic of alpha-amylase complex structures. Kinetic analysis, in comparison with HSAmy, revealed that the mutant exhibited a 70-fold decrease in the specific activity for starch and significantly reduced k(cat) (20-fold) and K(m) (4-fold) for maltoheptaoside. Collectively, these results suggest that W641 and the chain of water molecules may be critical for the alpha-amylase activity.

Introducing transglycosylation activity into human salivary alpha-amylase (HSA).

Synthesis of 4-nitrophenyl 1-thio-beta-D-maltoside, maltotrioside, and maltotetraoside in yields up to 60% has been achieved by a Tyr151Met (Y151M) mutant of human salivary alpha-amylase. Y151M is capable of transferring maltose and maltotriose residues from a maltotetraose donor onto different p-nitrophenyl glycosides. (1)H and (13)C NMR studies revealed that the mutated enzyme preserved the stereo- and regioselectivity. The glycosylation took place at position 4 of the glycosyl acceptor, forming the alpha(1-4)glycosidic bond, exclusively. [reaction: see text]

Detachment of Actinobacillus actinomycetemcomitans biofilm cells by an endogenous beta-hexosaminidase activity.

When cultured in broth, fresh clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans form tenaciously adherent biofilm colonies on surfaces such as plastic and glass. These biofilm colonies release adherent cells into the medium, and the released cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. We mutagenized A. actinomycetemcomitans clinical strain CU1000 with transposon IS903phikan and isolated a transposon insertion mutant that formed biofilm colonies which were tightly adherent to surfaces but which lacked the ability to release cells into the medium and disperse. The transposon insertion in the mutant strain mapped to a gene, designated dspB, that was predicted to encode a secreted protein homologous to the catalytic domain of the family 20 glycosyl hydrolases. A plasmid carrying a wild-type dspB gene restored the ability of biofilm colonies of the mutant strain to disperse. We expressed A. actinomycetemcomitans DspB protein engineered to contain a hexahistidine metal-binding site at its C terminus in Escherichia coli and purified the protein by using Ni affinity chromatography. Substrate specificity studies performed with monosaccharides labeled with 4-nitrophenyl groups showed that DspB hydrolyzed the 1-->4 glycosidic bond of beta-substituted N-acetylglucosamine, which is consistent with the known functions of other family 20 glycosyl hydrolases. When added to culture medium, purified DspB protein, but not heat-inactivated DspB, restored the ability of the mutant strain to release cells and disperse. DspB protein also caused the detachment of cells from preformed biofilm colonies of strain CU1000 grown attached to plastic and the disaggregation of highly autoaggregated clumps of CU1000 cells in solution. We concluded that dspB encodes a soluble beta-N-acetylglucosaminidase that causes detachment and dispersion of A. actinomycetemcomitans biofilm cells.

Subsite mapping of human salivary alpha-amylase and the mutant Y151M.

This study characterizes the substrate-binding sites of human salivary alpha-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).

Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase.

Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site.

The mechanism of salivary amylase hydrolysis: role of residues at subsite S2'.

Hydrolysis of starch or oligosaccharides by mammalian amylases, in general, results in maltose as the leaving group. The active site of these amylases harbors three aromatic residues Trp59, Tyr62, and Tyr151, which provide stacking interactions to the bound glucose moieties. We hypothesized that Tyr151, located at the S2' subsite, may influence the size of the leaving group. Therefore, using a baculovirus expression system, we generated a mutant Y151M in which the tyrosine at position 151 of human salivary amylase is replaced by a methionine. The specific activity, K(m), rate of hydrolysis, and the product distribution for Y151M were distinctly different from those of the wild-type enzyme using starch and oligosaccharides as substrates. The mutant enzyme Y151M consistently produced glucose as the minimal leaving group and exhibited a twofold increase in K(m). These results suggest that the stacking interaction at subsite S2' in the wild type plays a role in hydrolysis.

Expression, characterization, and biochemical properties of recombinant human salivary amylase.

Human salivary amylase, a major component of human salivary secretions, possesses multiple functions in the oral cavity. It is the only enzyme in saliva capable of degrading oligosaccharides, which are used by the oral microflora for nutritional purposes. In order to understand its role in disease processes such as caries, we have undertaken the structure-function analyses of amylase. In this regard, the nonglycosylated human salivary amylase was expressed in a baculovirus expression system. The native and the recombinant amylases exhibit similar biochemical as well as biophysical properties. Unlike recombinant human pancreatic amylase, recombinant human salivary amylase is not glycosylated when expressed in a baculovirus system as determined from the crystal structure determination of the recombinant enzyme. Therefore, this system is suitable for further structure-function work without resorting to enzymatic removal of the carbohydrate chain. Details of the expression, purification, and biophysical properties will be presented.