An active-site peptide containing the second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modifications

Abstract

The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and glycine ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained. Though 30 mM diethyl pyrocarbonate (DEP) also inactivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was modified by DEP, 50% of the activity was still retained. The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC. However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivation of the sucrose-cleaving activity. In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group. Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE. A fluorescent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala.(ABSTRACT TRUNCATED AT 250 WORDS)

A circularly permuted α‐amylase‐type α/β‐barrel structure in glucan‐synthesizing glucosyltransferases

“…Experimental evidence supports the idea that sites for sucrose cleavage and glucosyl transfer are distinct [2,47]. Thus in an L. mesenteroides dextran sucrase the sequence immediately preceding the predicted H3 has been implicated in sucrose binding and cleavage [12], while in glucosyltransferases the C-terminal region is recognised as a dextranbinding domain and a segment of ca. 300 residues following E3 is necessary for full transferase activity [10,[48][49][50].…”

“…Information is lacking on their threedimensional structure, but short stretches of sequence similarities exist [10][11][12] between the glucosyltransferases and the aamylases and related amylolytic enzymes that belong to a superfamily of multidomain proteins with a catalytic (fl/c08-barrel domain [13][14][15][16][17][18][19][20][21][22]. These barrel domains contain a few invariant residues involved in the enzyme mechanism and also flstrand-associated sequence motifs which provide a fingerprint of the enzyme origin and specificity [1 623].…”

Three-dimensional modelling of the catalytic domain of Streptococcus mutans glucosyltransferase GtfB

“…Primary sequence alignment revealed that this nanopeptide is highly conserved in the amylases and K-glucosidases [13]. A second active site identi¢ed by Funane et al [14] and by our group [13] that two Asp residues in Gtf-P1 are essential for sucrase activity but may play di¡erent roles in GtfB and -C [15]. Taken together, the N-terminal one-third of the GTFs may play a central role in sucrose splitting and glucan synthesis.…”

Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

“…Glucansucrase activity was assayed colorimetrically by the Nelson-Somogyi procedure 20) by measuring the release of reducing sugar from sucrose with glucose as a standard, as described previously. 21) One unit of dextransucrase releases 1 mole of reducing sugar from sucrose per min. Protein concentration was measured using a BCA protein assay reagent (Pierce) with bovine serum albumin as the standard.…”

“…2, ), containing the region ''LLANDVNSNP'' reported in L. mesenteroides. 21,24) The amino acid sequences of Site 1 are conserved in glucansucrases with about 50 to 70% identity, but there are no significant similar amino acid sequences in other proteins even in closely related family 13 enzymes. The identity of amino acid sequence of Site 1 of DSRS and DSRT5 is 55%.…”