The proposed research is a continuation of the study of the properties and mechanism of action of glucansucrases, Leuconostoc mesenteroides B- 512F dextransucrase, Streptococcus mutans 6715 dextransucrase (glucosyltransferase-soluble [GTF-S]) and S. mutans mutansucrase (glucosyltransferase-insoluble [GTF-I]). The study will focus on five aspects: (1) The study of the sucrose binding specificity. Sucrose analogues with the hydroxyl group at C-1', -6', and -2 replaced by H, F, and NH2 will be synthesized and studied as inhibitors or substrates for dextran synthesis. (2) The study of acceptor binding specificity. Analogues of alpha-methyl-D-glucopyranoside and 1,5-anhydro-maltitol in which the C-6-OH is replaced by H, F, and NH2 and alpha-Me-Glc in which the OH at C-2, -3, and -4 are replaced by H or inverted will be synthesized and studied as inhibitors of glucan synthesis and the structures of any resulting acceptor products determined. (3) The synthesis and study of 1-alpha-(haloacetyl)-D-glucopyranosides as specific active-site-directed inhibitors that alkylate the catalytic imidazole groups. (4) The study of the effect of acceptor reactions on dextran synthesis and the formation of branch linkages. Studies will be conducted on (a) the effect of the concentration of maltose; (b) the effect of viscosity on the structure of dextran synthesis; (c) the minimum size of isomaltodextrin required for the formation of a branch linkage; and (d) the reaction of GTF-I and GTF-S with maltodextrins DP 8-12 and with starch. (5) Study of the active site and the mechanism of action. Three experiments are proposed: (a) the determination of the number of sucrose binding-sites per enzyme molecule by equilibrium dialysis and equilibrium gel-filtration using [14C]-6-deoxy sucrose; (b) the determination of the number of acceptor binding-sites using the same techniques with [3H]-alpha-methyl-D-glucopyranoside and with [14C]maltose; and (c) the determination of the ratio of the acceptor reaction to the glycan synthesis as a function of partial inactivation by diethylpyrocarbonate and by dye photo-oxidation. The health significance involves a better understanding of the mechanism of glucansucrases and the formation of dental plaque by understanding: the substrate and acceptor binding specificity and thereby the development of inhibitors and the effect of acceptors and viscosity on dextran and plaque structure.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE003578-21
Application #
2128905
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-08-01
Project End
1996-09-29
Budget Start
1994-09-30
Budget End
1995-09-29
Support Year
21
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Iowa State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Ames
State
IA
Country
United States
Zip Code
50011
Su, D; Robyt, J F (1994) Determination of the number of sucrose and acceptor binding sites for Leuconostoc mesenteroides B-512FM dextransucrase, and the confirmation of the two-site mechanism for dextran synthesis. Arch Biochem Biophys 308:471-6
Kim, D; Robyt, J F (1994) Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase. Enzyme Microb Technol 16:1010-5
Kim, D; Robyt, J F (1994) Production and selection of mutants of Leuconostoc mesenteroides constitutive for glucansucrases. Enzyme Microb Technol 16:659-64
Tanriseven, A; Robyt, J F (1993) Interpretation of dextransucrase inhibition at high sucrose concentrations. Carbohydr Res 245:97-104
Su, D; Robyt, J F (1993) Control of the synthesis of dextran and acceptor-products by Leuconostoc mesenteroides B-512FM dextransucrase. Carbohydr Res 248:339-48
Fu, D T; Robyt, J F (1991) Maltodextrin acceptor reactions of Streptococcus mutans 6715 glucosyltransferases. Carbohydr Res 217:201-11
Fu, D T; Robyt, J F (1990) A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase. Prep Biochem 20:93-106
Fu, D T; Robyt, J F (1990) Acceptor reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase. Arch Biochem Biophys 283:379-87
Fu, D T; Slodki, M E; Robyt, J F (1990) Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydro Arch Biochem Biophys 276:460-5
Tanriseven, A; Robyt, J F (1989) Synthesis of 4,6-dideoxysucrose, and inhibition studies of Leuconostoc and Streptococcus D-glucansucrases with deoxy and chloro derivatives of sucrose modified at carbon atoms 3, 4, and 6. Carbohydr Res 186:87-94

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