CHARACTERIZATION OF LEUCONOSTOC-MESENTEROIDES NRRL B-512F DEXTRANSUCRASE (DSRS) AND IDENTIFICATION OF AMINO-ACID-RESIDUES PLAYING A KEY ROLE IN ENZYME-ACTIVITY

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran fr om sucrose or oligosaccharides when acceptor molecules, like maltose, are present, The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found t o be similar to the characteristics of the extracellular dextransucras e produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with gluco syltransferase sequences and four amino acid residues were selected fo r site-directed mutagenesis experiments: aspartic acid 511, aspartic a cid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp -551 were independently replaced with asparagine and His-661 with argi nine. Mutation at Asp-511 and Asp-551 completely suppressed dextran an d oligosaccharide synthesis activities, showing that at least two carb oxyl groups (Asp-511 and Asp-551) are essential for the catalysis proc ess. However, glucanbinding properties were retained, showing that DSR S has a two-domain structure like other glucosyltransferases. Mutation s at Asp-513 and His-661 resulted in greatly reduced dextransucrase ac tivity. According to amino acid sequence alignments of glucosyltransfe rases, a-amylases or cyclodextrin glucanotransferases, His-661 may hav e a hydrogen-bonding function.