Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase fromLeuconostoc mesenteroidesNRRL B-1355

Abstract

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.

Structure–function relationships of family GH70 glucansucrase and 4,6-α-glucanotransferase enzymes, and their evolutionary relationships with family GH13 enzymes

“…Several other strains, i.e. Leuconostoc mesenteroides NRRL B-1355 (Table 1 ) [ 29 ] and Streptococcus sobrinus were also found to contain multiple GSs [ 30 ]. These multiple GS enzymes generally display different product (linkage) specificity (Table 1 ), but it has remained unclear whether they have different physiological roles.…”

“…DSR-DP and DSR-M mainly catalyze the synthesis of α-glucans with (α1 → 6) linkages, while BRS-A introduces (α1 → 2) branching linkages [ 28 ]. Interestingly, L. mesenteroides NRRL B-1355 produces a GS (ASR) (Table 1 ) [ 29 , 40 ], synthesizing a glucan with alternating (α1 → 6) and (α1 → 3) linkages [ 40 ]. This distinct α-glucan has been named alternan and its enzyme was designated alternansucrase (ASR, EC 2.4.1.140).…”

Structure and Function of Glucansucrases

“…Sequence repeats A and C (see Table 1) are found in Streptococcus and Leuconostoc species [7]. Other sequence repeats unique to L. mesenteroides ASR [10] and L. parabuchneri GTF33 [15], respectively, have also been identified (Table 1). Other sequence repeats unique to L. mesenteroides ASR [10] and L. parabuchneri GTF33 [15], respectively, have also been identified (Table 1).…”

“…It alternates the formation of α(1→3) and α(1→6) bonds, and therefore the synthesized αglucan is named alternan [10]. It alternates the formation of α(1→3) and α(1→6) bonds, and therefore the synthesized αglucan is named alternan [10].…”

Structural determinants of alternating (α1 → 4) and (α1 → 6) linkage specificity in reuteransucrase of Lactobacillus reuteri

“…On the other hand, most of the α-glucans produced by Streptococcus strains are found to predominantly contain (α1 → 3) linkages, and are named mutans; they are generally insoluble and have been recognized as an important pathogenic factor in the development of dental caries 5 8 9 10 11 17 18 19 20 . L. mesenteroides NRRL B-1355 produces a special glucansucrase ASR, which synthesizes alternan with alternating (α1 → 6) and (α1 → 3) linkages 21 22 , and is incapable of synthesizing two consecutive (α1 → 3) linkages 23 . The alternation of (α1 → 6) and (α1 → 3) linkages in alternan makes this polysaccharide resistant to endodextranase hydrolysis and is considered to be the determinant for its distinct physical properties 24 .…”