Introduction
Dental plaque is the accumulation of biofilm, a collection of various microbial species that colonize the surfaces of our teeth. This diverse set of microorganisms include things like bacteria and fungi, and believe it or not, it is actually perfectly normal and even advantageous to have these things living inside our mouths. This is because some of the microorganisms that make up our biofilm are considered favorable or “good” in that they prevent colonization of things like the “bad,” not so favorable microorganisms – an idea of commensalism that helps us defend ourselves against harmful, infectious microbial species. Although at times beneficial to our oral health, some of the microorganisms that live within these oral biofilms can, if given the opportunity to, over grow within the mouth and become pathogenic, having the ability to damage the teeth and gums and potentially cause an oral infection and/or disease. This is why dental plaque control is extremely important to not only our oral health, but systemic health too. The question is, however, if we want to figure out how to control the formation of dental plaque, we need to understand how biofilms are made. Only then can we begin to find an alternative to fight against its accumulation and prevent oral disease.
One of the many contributing factors to the formation of dental plaque is complex, extracellular sugars such as (1→3)-α-glucans produced by bacterial and fungal species living and growing inside the mouth. (1→3)-α-glucans are insoluble, linear α-1,3-linked homopolymers of D-glucose that play a key role in the structure and function of the biofilm matrix. (1→3)-α-glucans possess (1→3)-α-glycosidic bonds and hydrogen bonds that provide the sugar with rigidity and water insolubility, respectively. This not only allows (1→3)-α-glucans to remain undissolved in the oral cavity, but it also helps them resist being washed away with oral fluids [1]. The (1→3)-α-glycosidic bonds of (1→3)-α-glucans are also resistant to enzymes naturally present in the oral cavity, and this enables the sugar to form and maintain a stable, durable biofilm matrix that can accumulate around the teeth as dental plaque [2][3]. However, there are enzymes, known as (1→3)-α-glucanases, that can be introduced into the oral cavity to specifically target and break down these sugars. (1→3)-α-glucanases are a type of glycosidase, or glycoside hydrolase, meaning that they work by catalyzing the hydrolysis of the (1→3)-α-glycosidic bonds in (1→3)-α-glucans. Knowing their function, we can use these hydrolytic enzymes in oral hygiene products, such as toothpaste and mouthwashes, to fight against bacterial and fungal (1→3)-α-glucans and the sugars’ contribution to the structure and function of the biofilm matrix. Perhaps introducing these enzymes to the oral cavity would essentially reduce the formation of dental plaque, and therefore, prevent the development of infectious oral diseases.
Molecular Biology of (1→3)-α-Glucanases
Unlike other glycoside hydrolases such as amylases, cellulases, chitinases, and even β-glucanases for that matter, there is not much biochemical and structural information on (1→3)-α-glucanases. For the most part, (1→3)-α-glucanases are generally known; however, we do not have enough information on the enzymes to understand the specifics. Although this makes it more difficult for us to thoroughly explore the enzymes’ structural components such as shape, what residues make up the active site, as well as additional residues or cofactors that may be significant to the enzymes’ stability or mode of action, we do have some basic knowledge on the molecular biology of (1→3)-α-glucanases.
(1→3)-α-glucanases are naturally produced by various fungal and bacterial species. For this reason, (1→3)-α-glucanases can be classified into two families of glycoside hydrolases, GH-71 and GH-87. Although this classification was originally made based on differences in amino acid sequence, the two families differentiate between which microbial species the enzymes are from. While GH-71 (1→3)-α-glucanases belong to a family of enzymes found in fungal species (1→3)-α-glucans, GH-87 (1→3)-α-glucanases are those that are made by bacteria. The two families also differ in molecular weights, a finding that has been determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE). While fungal enzymes range from a limited 67-90 kDa, bacterial enzymes vary from a much larger range of 48-160 kDa. Most (1→3)-α-glucanases, regardless of family origin, have been identified as monomeric proteins; however, there have been reports suggesting that the enzymes could also be dimers or even tetramers [4].
With the use of nucleotide and amino acid sequencing, bacterial and fungal genes that encode proteins with (1→3)-α-glucanase activity have been identified. Based on these analyses, both bacterial and fungal genes show significant sequence identity, sharing high homology among strains of the same species. In bacterial enzymes, (1→3)-α-glucanases have a C-terminal domain that is considered the catalytic domain, as this domain holds the primary glucanase activity. The N-terminal domain, in contrast, is more supportive in that it contains additional supplementary modules that are involved in substrate binding. Although this kind of organization is also found in fungal (1→3)-α-glucanases, the terminal domains of these enzymes differ in responsibility. In fact, functions of the terminal domains found in fungal (1→3)-α-glucanases are quite opposite from those of bacterial enzymes. In fungal (1→3)-α-glucanases, the C-terminal domain rather functions as the substrate binding domain while the N-terminal domain is the catalytic domain. This is because, unlike in bacterial (1→3)-α-glucanases, the C-terminal domain is what targets the enzyme to the sugar substrate and the N-terminal domain, on the other hand, works by enzymatically cleaving the glycosidic bonds [4].
Characterization of (1→3)-α-Glucanases
Numerous (1→3)-α-glucanases have been purified and characterized, giving us insight into their catalytic behavior and stability when exposed to certain temperature and pH conditions. Through extensive experimentation, the optimal temperature for both bacterial and fungal (1→3)-α-glucanases was found to range from 40 to 56 ℃. However, when it comes to optimal pH, the two families differ quite drastically. While fungal enzymes have increased activity in acidic environments with a pH range of 4.5 to 5.5, bacterial enzymes work best in a more neutral environment with slightly higher pH values of 5.5 to 6.9. This is because fungal (1→3)-α-glucanases are only stable at a limited pH range of 5.0 to 7.0, whereas bacterial (1→3)-α-glucanases maintain stability at a much larger pH range that includes alkaline conditions, ranging from 4.0 to 11.0 [4].
All (1→3)-α-glucanases function as glycoside hydrolases that break down the glycosidic bonds of (1→3)-α-glucans. Some of these enzymes are thought to function through a processive mechanism, meaning that they remain attached to their substrate, in this case (1→3)-α-glucans, and repetitively catalyze the hydrolysis of the sugar’s glycosidic bonds before dissociating [5]. However, the way that these enzymes go about doing so can be different from one another in terms of starting point of hydrolysis and what products are released. We differentiate between these modes of action by organizing (1→3)-α-glucanases into two distinct groups, exo-(1→3)-α-glucanases and endo-(1→3)-α-glucanases. Exo-(1→3)-α-glucanases hydrolyze terminal (1→3)-α-glucoside linkages at non-reducing ends and release monosaccharides and disaccharides. In contrast, endo-(1→3)-α-glucanases hydrolyze internal (1→3)-α-glucoside linkages at random sites and release nigerooligosaccharides. Although the exo-(1→3)-α-glucanases and endo-(1→3)-α-glucanases act at different locations on the (1→3)-α-glucan chain and release varying products, their catalytic function is the same in that they both hydrolyze the (1→3)-α-glycosidic bonds of (1→3)-α-glucans, perhaps through a processive mechanism [4].
Additionally, all (1→3)-α-glucanases have a high specificity for their substrate, (1→3)-α-glycosidic linkages, making them extremely effective in catalyzing the degradation of (1→3)-α-glucan sugars. In fact, some of these enzymes even hydrolyze (1→3)-α-glucan sugars that include other linkages such as (1→4)-α-linkages and (1→6)-α-linkages in addition to (1→3)-α-linkages. This means that (1→3)-α-glucanases can not only break down linear (1→3)-α-glucans, but also branched (1→3)-α-glucans, as both forms of these sugars include their (1→3)-α-glycosidic linkage substrate [6][7].
Examples of (1→3)-α-Glucanases
Again, (1→3)-α-glucanas are extracellular sugars that can be made by bacteria or fungi, which explains why there are (1→3)-α-glucanases for both families. Let us look at a (1→3)-α-glucanase from a Streptococcus species, Streptomyces thermodiastaticus strain HF3-3 (Agl-ST). Since this is a bacterial species, the (1→3)-α-glucanase has been identified as a glycoside hydrolase family 87 (GH87). This particular enzyme is also classified as an endo-(1→3)-α-glucanases, as it specifically hydrolyzes Streptococcal (1→3)-α-glucans’ internal glycosidic bonds at random sites along the chain. The enzyme has a sequence length of 610 and is considered a dimer, having its structure consist of two protein domains. Its monomeric weighs 126.87 kDa and is one single protein chain composed of two modules, a and a [8].
This type of structure is also found in another (1→3)-α-glucanase member from the glycoside hydrolase family 87 (GH87), Paenibacillus glycanilyticus strain FH11 (Agl-FH1) - with moderate sequence identities between each other (approximately 27% between the catalytic units) [8]. Just as in Streptomyces thermodiastaticus strain HF3-3, Paenibacillus glycanilyticus strain FH11 hydrolyzes Streptococcal (1→3)-α-glucans’ glycosidic bonds with an endo mode of action. In contrast, this enzyme only consists of one single protein chain, making it a monomer with a shorter sequence length of 573. This makes sense as to why its catalytic domain is also much smaller in size, only weighing 63.33 kDa. The crystal structure of this enzyme has been identified, revealing the Agl-FH1 to resemble that of Agl-ST, also being made up of two modules, the and the [9].
Conclusion
Since sugars such as (1→3)-α-glucans are a primary component of the biofilm matrix and partly responsible for the formation of dental plaque, it makes them potential targets for antimicrobial agents. By including the hydrolytic enzymes (1→3)-α-glucanases into oral hygiene products, like toothpaste and mouthwashes, we have the opportunity to compromise the structural integrity and function of the biofilm matrix. This is because introducing these enzymes into the oral cavity would break down bacterial and fungal (1→3)-α-glucans by catalyzing the hydrolysis of their glycosidic bonds. This would essentially make the formation of dental plaque a more difficult task for bacteria and fungi that produce these sugars, and therefore, prevent the development of infectious oral diseases such as dental caries and gum disease. Not to mention, using enzymes like (1→3)-α-glucanases as an alternative to mechanically clean our mouths is hopeful and beneficial because, unlike most antiseptics, they are substrate specific. (1→3)-α-glucanases only target the (1→3)-α-glycosidic bonds of bacterial and fungal (1→3)-α-glucans, known components of the biofilm matrix, while disregarding other things like commensal microorganisms. Other antimicrobials that are used in our oral hygiene products, like alcohol for example, do not discriminate like this - they inhibit the bad microorganisms as well as the good microorganisms that are necessary to maintain our normal oral microbiota, giving them the potential to disrupt our natural balance of microorganisms in the mouth. This is why (1→3)-α-glucanases could be used as an alternative to control dental plaque and the development of infectious oral diseases, helping us maintain and improve our overall oral health.
