Conserved Repeat Motifs and Glucan Binding by Glucansucrases of Oral Streptococci and Leuconostoc mesenteroides

Abstract

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly ␣-1,3 or ␣-1,6 links, as well as alternating ␣-1,3 and ␣-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70

“…In particular, three-dimensional structures in complex with either isomalto-oligosaccharides or dextrans, the natural acceptors of these enzymes, were heavily lacking, thus preventing molecular descriptions of isomalto-oligosaccharide or dextran-binding sites. In particular, no complexes were previously disclosed in which carbohydrates were present in domain V, although this domain was biochemically proven to act as a glucan-binding domain in several GH70 enzymes (25,31,32) and was suggested to participate in the processivity of polymerization (26,28,29). Regarding the ␣-(132) branching sucrase (⌬N 123 -GBD-CD2) enzymes, structural data are even scarcer as only two structures in the apo form have been solved thus far (9).…”

“…In particular, the five repeats found at the C-terminal extremity of the GBD of DsrP glucansucrase are capable of binding the cell surface of Leuconostoc cells (24). Mutagenesis of conserved aromatic or basic residues of the repeats found in Streptococcus downei GtfI was performed and led to a reduction of the glucan binding capability (25). The elimination of some repeats by truncation was also shown to influence transglucosylation, modify glucan binding properties, and affect the polymer size (26 -29).…”

“…Then, the primary structure of the full-length GBD of DSR-E (57) was analyzed using the online repeat detection tool TRUST (58), and this allowed detecting 12 sequence repeats in this domain. Similarly, repeats corresponding to glucan binding pockets were identified by sequence analyses of domain V of several glucansucrases that were experimentally shown to bind ␣-glucans (namely GBD4RS of GTF-I from Streptococcus sobrinus, GBD1A of GTF-I from S. downei, and GBD5 of DSR-S from L. mesenteroides B-512F) (25,31,32). These repeats were then aligned using the web service Expresso (from T-coffee suite), providing structure-based multiple sequence alignment.…”

Role of the C-terminal region of dextransucrase from Leuconostoc mesenteroides IBT-PQ in cell anchoring

“…2a). Within these structural motifs, A repeats, usually present in these regions (Janecek et al, 2000;Shah et al, 2004), are found. Moreover, other repeat units found in this type of protein, such as IDGYYFD+N+G (von Eichel-Streiber et al, 1992) and the Y-G repeats (Giffard & Jacques, 1994), are also contained in these structural motifs (Fig.…”

“…This region is involved in binding to the glucan in streptococcal and lactobacilli GSs, and has therefore been designated the glucan binding domain (GBD) (Shah et al, 2004;Krajl et al, 2004a). However, there is controversy about its function in Leuconostoc dextransucrases, as although several authors report strong binding of the C-terminal region to dextran (Miller et al, 1986;Funane et al, 1998), other authors are not able to find this interaction (Shah et al, 2004).…”