Role of the C-terminal YG repeats of the primer-dependent streptococcal glucosyltransferase, GtfJ, in binding to dextran and mutan.
The recombinant primer-dependent glucosyltransferase GtfJ of Streptococcus salivarius possesses a C-terminal glucan-binding domain composed of eighteen 21 aa YG repeats. By engineering a series of C-terminal truncated proteins, the position at which truncation prevented further mutan synthesis was defined to a region of 43 aa, confirming that not all of the YG motifs were required for the formation of mutan by GtfJ. The role of the YG repeats in glucan binding was investigated in detail. Three proteins consisting of 3.8, 7.2 or 11.0 C-terminal YG repeats were expressed in Escherichia coli. Each of the three purified proteins bound to both the 1,6-alpha-linked glucose residues of dextran and the 1,3-alpha-linked glucose residues of mutan, indicating that a protein consisting of nothing but 3.8 YG repeats could attach to either substrate. Secondary structure predictions of the primary amino acid sequence suggested that 37% of the amino acids were capable of forming a structure such that five regions of beta-sheet were separated by regions capable of forming beta-turns and random coils. CD spectral analysis showed that the purified 3.8 YG protein possessed an unordered secondary structure with some evidence of possible beta-sheet formation and that the protein maintained this relatively unordered structure on binding to dextran.
Kingston
Kim B
KB
Institute of Dental Research, Centre for Oral Health, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia1.
Allen
Donna M
DM
Institute of Dental Research, Centre for Oral Health, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia1.
Jacques
Nicholas A
NA
Institute of Dental Research, Centre for Oral Health, Westmead Hospital, PO Box 533, Wentworthville, NSW 2145, Australia1.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D016384:Consensus Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D003911:Dextrans / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D003911:Dextrans; D005936:Glucans; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005964:Glucosyltransferases