Analysis of the Structure and Functional Regions of Dextransucrase from Leuconostoc mesenteroides

Abstract

Purified dextransucrase (EC 2.4.1.5) was obtained from a Leuconostoc mesenteroides NRRL B-5 12F culture supernatant by affinity chromatography with DEAE-Sephadex A-50 followed by Sepharose 6B gel filtration with a yield of about 25%. Chemical modifications with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), dietylpyrocarbonate (DEP), and o-phthalaldehyde (OPA) were done, respectively, on the purified dextransucrase. All three substances inactivated dextransucrase. Addition of the substrates sucrose and dextran retarded the enzyme inactivation by EDC and OPA but not by DEP. Differential labeling of dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphtyl)ethyle-nediamine (EDAN) was used as the nucleophile. A fluorescent-labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The isolated peptide was located at 30-45 amino acids toward the amino terminal from the catalytic aspartic acid and seemed to contain the second essential carboxyl group for the catalytic activity. Differential modification of dextransu-crase by OPA was conducted in the presence of sucrose, dextran, or sucrose-monocaprate. Modi-fied enzymes were digested by trypsin. The resultant peptides containing lysine residues protected by the ligands were homologous to the catalytic domain of dextransucrases and streptococcal glu-cosyltransferases (GTFs), but these regions were separate from the catalytic aspartic acid and the second essential carboxyl group. Four glucan-binding peptides of dextransucrase were obtained by mild trypsin digestion using mutan as the glucan in the presence of ammonium sulfate. Three pep-tides were located in the catalytic domain. One of them was identical with the dextran-binding pep-tide that contains lysine, which was isolated by differential chemical modification with OPA. Addi-tionally, there was a peptide similar in sequence to the glucan-binding A-repeat of dextransucrases and GTFs. A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of L. mesenteroides NRRL B-S 12F. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-S 12F, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. The dsrT gene product ex-pressed in E. coli BL21 (DE3) did not produce dextran. The insertion of five nucleotides at the pu-tative deletion point in dsrT added glucan-binding domain to the enzyme and its dextransucrase ac-tivity was restored. The glucan produced by five-nucleotide-inserted dsrT (dsrT5) was mainly water insoluble and contained about 40% of 1, 3-linkages by methylation analysis. L. mesenteroides NRRL B-512F dextran is water soluble and contains about 95% a -1, 6-linkages. The presence of the dsrT gene suggests that this strain originally produced the different structure of dextran.