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 alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-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.

FIG. 1.

Conservation of individual residues in the 33-amino-acid A repeat derived from CLUSTAL alignment. The consensus sequence is shown at the bottom.

FIG. 2.

Kinetics of binding of biotinylated dextran to the recombinant binding domain of S. downei GtfI (GBD0).

FIG. 3.

Competition with biotinylated dextran for binding to GBD0 by unlabeled dextran, isomaltosaccharides, and nigerooligosacharides.

FIG. 4.

Competition with biotin-dextran for the binding domain of S. downei GtfI (GBD0) by unlabeled dextran T70, choline, and alternan. The concentration of biotinylated dextran was kept constant at 1 mM (a) or 100 μg/ml (b). The competitors choline or alternan were added after a 10-min preincubation with biotin-dextran and washed after a further 10-min incubation. Detection and washing steps were carried out as described in Materials and Methods. In panel b, the units for the ligands are micrograms per milliliter because the molecular weight distribution of the alternan is unknown.

FIG. 5.

SDS-PAGE analysis of cell extracts from L. mesenteroides and S. mutans strains. (A) Zymogram of active glucansucrases after SDS-PAGE separation and incubation in sucrose. (B) The same samples electroblotted and developed with biotin-dextran to detect glucan binding proteins. Lane 1, L. mesenteroides NRRL B-1355; lane 2, L. mesenteroides NRRL B-512F; lane 3, L. mesenteroides NRRL B-1299; lane 4, S. mutans UA159; lane M, molecular mass ladder of 250, 150, and 100 kDa (arrows).

FIG. 6.

(a) A repeats present in recombinant GBD1A, which was derived by truncating the GBD of S. downei GtfI (41). Amino acid substitutions introduced by mutagenesis are indicated below the main sequence. (b) Relative binding of biotinylated dextran by GBD1A and specific mutant forms. YF-FY, residues 1314 and 1315.