Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran

Abstract

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

FIG. 1.

SDS-PAGE profiles of recombinant DSR-E and truncated forms produced by E. coli TOP10. Lanes: M, broad-range prestained precision protein standard (Bio-Rad); 1, DSR-E, 325 kDa; 2, Δ(VZ), 304 kDa; 3, Δ(CD2), 230 kDa; 4, CD1, 116 kDa; 5, GBD-CD2, 192 kDa; 6, CD2, 111 kDa.

FIG. 2.

Reverse-phase HPLC chromatograms of the oligosaccharides synthesized by the different crude truncated forms of DSR-E in the presence of sucrose and maltose. The enzymes used were (a) native L. mesenteroides NRRL B-1299 dextransucrase, (b) complete DSR-E, (c) DSR-E with the variable zone deleted [Δ(VZ)], (d) DSR-E with the second catalytic domain deleted [Δ(CD2)], (e) the first catalytic domain alone (CD1), (f) CD2 linked with GBD (GBD-CD2), and (g) the second catalytic domain alone (CD2). F, fructose; M, maltose; S, sucrose; P, panose, or α-d-glucopyranosyl-1,6-α-d-maltose; α-1,6 GOS DP4, α-d-glucopyranosyl-1,6-α-d-glucopyranosyl-1,6-α-d-maltose; α-1,6 GOS DP5, α-d-glucopyranosyl-1,6-α-d-glucopyranosyl-1,6-α-d-glucopyranosyl-1,6-α-d-maltose; α-1,2 GOS DP4, α-d-glucopyranosyl-1,2-α-d-glucopyranosyl-1,6-α-d-maltose; α-1,2 GOS DP5, α-d-glucopyranosyl-1,2-α-d-glucopyranosyl-1,6-α-d-glucopyranosyl-1,6-α-d-maltose. The profiles obtained with crude or micropurified enzyme are similar (not shown).

FIG. 3.

HPAEC chromatograms of the oligosaccharides synthesized by the truncated form GBD-CD2 of DSR-E in the presence of sucrose and α-1,6 GOS.

FIG. 4.

¹³C NMR analysis of the dextran synthesized by dextransucrases from L. mesenteroides NRRL B-512F before (A) and after (B) modification by the variant GBD-CD2 of DSR-E.