Shah Deepan S H, Russell Roy R B
School of Dental Sciences, University of Newcastle, Newcastle upon Tyne NE2 4BW, UK.
Microbiology (Reading). 2004 Jun;150(Pt 6):1947-1956. doi: 10.1099/mic.0.26955-0.
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 alpha/beta 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.
变形链球菌产生胞外葡糖基转移酶（GTFs），其可利用蔗糖合成葡聚糖。这些葡聚糖对于决定牙菌斑的渗透性和黏附性很重要。GTFs和GbpA葡聚糖结合蛋白的特征是具有一个包含一系列33个氨基酸重复序列的结合结构域，称为“A”重复序列。在变形链球菌基因组序列中搜索含有“A”重复序列的开放阅读框（ORF），并鉴定出一个新基因gbpD，该基因似乎是变形链球菌群所特有的。GbpD序列显示在蛋白质中部存在三个“A”重复序列，一种新的葡聚糖结合试验表明GbpD以2 - 3 nM的解离常数（K(D)）与葡聚糖结合。构建GbpD的截短衍生物证实“A”重复区域对于结合至关重要。此外，gbpD基因敲除突变体在由蔗糖衍生的聚合物诱导的聚集程度上有所改变。GbpD的N端有一个信号序列，其后是在公共数据库中无同源物的区域，而C端与α/β水解酶家族（包括脂肪酶和羧酸酯酶）具有同源性。GbpD含有这些酶的两个典型区域：一个GxSxG活性位点“脂肪酶盒”和一个“氧阴离子洞”。在有钙存在的情况下，GbpD从一系列甘油三酯中释放出游离脂肪酸（FFA），表明具有脂肪酶活性。葡聚糖结合/脂肪酶双功能表明该酶的天然底物可能是由与脂质相连的碳水化合物组成的表面大分子。gbpD突变体的疏水性低于野生型，并且纯重组GbpD降低了变形链球菌和另一种牙菌斑细菌血链球菌的疏水性。GbpD与血链球菌的脂磷壁酸（LTA）结合并从其中释放FFA，但对变形链球菌的LTA没有影响。这些结果引发了一个有趣的可能性，即GbpD可能参与牙菌斑生物膜内的直接种间竞争。
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