Glucansucrase

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Introduction

Glucansucrase is mainly produced by Leuconostoc mesenteroides and oral Streptococcus species, and lactic acid bacteria (Lactococci, Lactobacilli) can also be produced. They are responsible for synthesizing sucrose into high molecular weight D-glucose polymers called glucans. The synthesis of low molecular weight oligosaccharides can also be catalyzed in the presence of efficient acceptors. The synthesis of glycosidic bonds does not require the mediation of nucleotide-activated sugars and cofactors. Glucansucrases are of industrial value due to their ability to produce glucans and oligosaccharides, and biologically important due to their critical role in cariogenic processes.

Glucansucrases were first discovered more than 50 years ago, but their mechanism of action is still not fully understood. In order to synthesize oligosaccharides of biological interest or develop vaccines against dental caries, it is important to elucidate the factors that determine the regiospecificity and regioselectivity of glucansucrases. In addition to structure-function relationship studies, cloning of the gene encoding glucansucrase has allowed us to identify important amino acid residues and found that the enzyme consists of two functional domains: a core region involved in sucrose binding and cleavage (~1000 amino acids) and a C-terminal domain (~500 amino acids) consisting of tandem repeats involved in glucan binding. Enzymatic studies have proposed different models for their mechanism of action. The use of secondary structure prediction has led to a clearer understanding of the structure-function relationship of glucansucrases.

Glucansucrases

Glucanucrase (EC 2.4.1.5) is an extracellular enzyme mainly produced by the soil bacterium Leuconostoc mesenteroides (commonly termed dextransucrase), Streptococcus species from the oral fora (commonly termed glucosyltransferases) and by lactic bacteria Lactococci. They are responsible for catalyzing the synthesis of high molecular weight D-glucose polymers from sucrose, and when efficient acceptors such as maltose or isomaltose are added to the reaction medium, glucansucrases catalyze the production of low molecular weight oligosaccharides rather than high molecular weight glucan.

Glucansucrases from L. mesenteroides are commonly used in industrial production. Dextran produced by L. mesenteroides NRRL B-512F was one of the first biopolymers produced on an industrial scale in 1948 and has found a variety of applications in medicine, separation technology and biotechnology. L. mesenteroides NRRL B-1299 produces oligosaccharides with one or more D-glucopyranosyl units linked through α(1-2) glycosidic bonds, which are highly resistant to attack by digestive enzymes and can be used as prebiotics in cosmetic and human nutritional applications. Glucansucrase produced by oral streptococci plays an important role in the cariogenesis process because the produced glucan enhances the attachment and colonization of cariogenic bacteria. In order to develop vaccines against dental caries, a great deal of effort has been devoted to the study of the genes encoding these enzymes. The sequences of 14 different glucansucrase encoding genes (named gtf) are now available.

Figure 1. Reactions catalysed by glucansucrases (Monchois, V., et al. 1999)

Structural and functional organization of the Glucansucrases

Studies initiated many years ago isolated and sequenced 17 genes encoding glucansucrases produced by oral streptococci involved in the cariogenesis process, S. mutans GS5 or LM7, S. sobrinus serotype h (S. downei Mfe28), serotype g (S. sobrinus 6715), serotype d (S. sobrinus OMZ176), by S. salivarius ATCC 25975 and by L. mesenteroides NRRL B-512F and B-1299. Isolated glucanases are all large enzymes with an average molecular weight of 160 000. Streptococci glucansucrase mainly synthesizes α(1-3)-rich mutated polysaccharides and α(1-6)-rich dextran polysaccharides. L. mesenteroides glucansucrase produces α(1-6)-rich dextran. However, genes encoding for glucansucrases-producing branched glucan through α(1-2) or α(1-4) glycosidic bond or synthesizing alternan have not been isolated. Studies of the analysis and comparison of different protein sequences have shown that these enzymes are closely related and share a common structure. They consist of four distinct domains. With the exception of DSR-A from L. mesenteroides NRRL B-1299, they all start at the N-terminal end begins with a 32-34 amino acid signal peptide followed by a highly variable stretch of 123-129 amino acids. They present a highly conserved core region of approximately 1000 amino acids, also known as the catalytic domain, which is a sucrose-binding domain capable of binding and cleaving sucrose. The C-terminal end of glucansucrase covers about 500 amino acids and consists of a series of tandem repeats that constitute the glucan-binding domain.

Figure 2. Schematic structure of glucansucrases for which encoding genes have been cloned. A, signal peptide; B, variable region; C, N-terminal catalytic domain; D, C-terminal glucan binding domain (Monchois, V., et al. 1999)