A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans

School of Dental Sciences, University of Newcastle, Newcastle upon Tyne NE2 4BW, UK Correspondence Roy R. B. Russell r.r.russell{at}ncl.ac.uk Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called ‘A’ repeats. The S. mutans genome sequence was searched for ORFs containing ‘A’ repeats, and one novel gene, gbpD , which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three ‘A’ repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K D of 2–3 nM. Construction of truncated derivatives of GbpD confirmed that the ‘A’ repeat region was essential for binding. Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the / hydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site ‘lipase box’ and an ‘oxyanion hole’. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S. mutans and another plaque bacterium, Streptococcus sanguinis . GbpD bound to and released FFA from lipoteichoic acid (LTA) of S. sanguinis , but had no effect on LTA from S. mutans . These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm. Abbreviations: FFA, free fatty acid; GBD, glucan-binding domain; Gbps, glucan-binding proteins; GTF, glucosyltransferase; LTA, lipoteichoic acid; 4MU, 4-methylumbelliferone; PHB, polyhydroxybutyrate; PHBd, polyhydroxybutyrate depolymerase