Mechanism of Streptococcus mutans glucosyltransferases: hybrid-enzyme analysis.
Streptococcus mutans GS5 expresses three glucosyltransferases (GTFs): GTF-I and GTF-SI, which synthesize water-insoluble glucans in a primer-independent manner, and GTF-S, which is responsible for the formation of primer-dependent soluble glucan. The amino acid sequences of the GTF-I and GTF-S enzymes exhibit approximately 50% sequence identity. Various hybrid genes were constructed from the structural genes for the enzymes, and their products were analyzed. Three different approaches were used to construct the hybrid enzymes: (i) ligation of DNA fragments containing compatible endonuclease restriction sites of the two genes at homologous positions; (ii) in vivo recombination between the homologous regions of each gene; and (iii) random fusion of DNA fragments from each gene generated following exonuclease III digestion of tandemly arranged fragments corresponding to the two functional domains of each enzyme. Hybrid GTFs composed of the sucrose-binding domain of one enzyme (GTF-I or GTF-S) with the glucan-binding domain of the other synthesized insoluble glucan exclusively in the absence of primer dextran. Insoluble glucan synthesis by some, but not all, of the GTF-S:GTF-I chimeric enzymes was stimulated by primer dextran T10 addition. In addition, glucan binding by the former but not latter group of hybrid GTFs was demonstrated. These results suggest that the glucan-binding domain alone does not solely determine primer dependence or independence or the structure of the resulting glucan product, although this carboxyl-terminal domain containing direct repeating units does appear to play a significant role in primer dependence.
Nakano
Y J
YJ
Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio 78284.
Kuramitsu
H K
HK
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D013295:Streptococcus mutans / Q000201:enzymology*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases