The data reported here describe the cloning of a new gene, named dsrE, obtained from L. mesenteroides strain NRRL B-1299. As confirmed by glucan synthesis and periodic acid-Schiff activity staining, cultures of E. coli clones harboring the plasmid carrying the dsrE gene produced an active dextransucrase. Recombinant DSR-E was demonstrated to produce, with maltose as the acceptor, ODi and Ri oligosaccharides which contain α-1,2 linkages.
In addition to its unique regiospecificity, DSR-E possesses a very original structure, never observed before, characterized by the presence of an additional catalytic domain at the carboxy terminus. With a calculated molecular mass of 313,267 Da, DSR-E has twice the average mass of GTFs and DSRs (32
The role and significance of the variable nonconserved region located downstream of the signal peptide remain unclear. Several studies showed that its deletion does not affect the enzyme activity (1
). However, in DSR-E we observed the presence of a 14-amino-acid repeated unit never identified before. That repeat, named S, could thus play a possible role in the enzymatic activity and/or specificity of DSR-E. Similarly, some glucansucrases with unusual specificities, for example, alternansucrase and DSR-T, also possess interesting repeats in the variable domain. So, the influence of this particular repeat on the catalytic properties of DSR-E must be evaluated with deletion experiments.
DSR-E is also remarkable because of the presence of a long GBD, ensuring the junction between the two catalytic domains CD1 and CD2. Along the GBD sequence, regularly alternating A and C repeats are found in which three CW units can be localized. From these observations, it can be suggested that the structure of GBD resembles that of the C-terminal choline binding domain of the autolysin LytA from S. pneumoniae
. Thus, A-C tandem repeats could be due to the recurrence of specific duplication events of an ancestral CW unit triplet, corresponding to a complete turn of an original superhelix. This hypothesis corroborates (i) the initial suggestions of Giffard and Jacques (17
), who proposed a definition of a fundamental repeating unit from which all classes of repeats (A, B, C, and D) are derived, and (ii) several studies that describe the presence of tandem repeats (4
Attention has been focused recently on repeated elements in the variable one-third of glucansucrases from L. mesenteroides
). Unlike DSR-S, DSR-B, and ASR (alternansucrase), no A and C motifs can be found in this region. However, DSR-E is the very first and sole glucansucrase in which a catalytic domain is located after the GBD. Such a structure can be related to the presence of repeated units upstream of the catalytic domain in other DSRs. Either DSR-E might be the product of gene fusion caused by the recombination of two dsr
genes, or recombination events between two dsrE
genes have led to the presence of repeated elements usually found in the GBD in the N terminus of glucansucrases from L. mesenteroides
The third and fifth domains correspond to two potential catalytic domains (CD1 and CD2), conferring to DSR-E a unique structure never observed before by analogy to glycoside hydrolase family 70 enzymes. Both domains cover about 900 amino acids, as in other GTFs (32
), and thus, because of the presence of all the amino acids thought to play key roles in catalysis, DSR-E seems to possess a double catalysis system. CD1 and CD2 share 44% identity with each other and an average identity with other GTF and DSR catalytic domains of 53 and 44% for CD1 and CD2, respectively. The lower similarity of CD2 can be explained by several regions that diverge from consensus sequences. A tryptophan residue at position 2135 stands for a usually conserved histidine residue. This amino acid is thought to play a role in glucan and oligosaccharide binding (35
). Peptide 2210
DAVDFIHNDTIQR in block C, the block containing the putative nucleophile, is very different from the highly conserved DAVDNVDADLLQI peptide found in all GTFs and DSRs. The usually conserved residues located just downstream of the first catalytic Asp in glucansucrases could constitute part of the subsite +1, which is involved in the acceptor binding (27
). The structure of this site determines the positioning of the acceptor molecule and thus the type of glucosidic bond formed. All glucansucrase enzymes from family 70 have an Asn residue at a position equivalent to N555 of DSR-S. In CD2, the corresponding dipeptide NV is replaced with 2214
FI, which can also be found in the amylosucrase sequence. Structural data obtained for this enzyme (52
) suggest that the Phe residue could be engaged in the specificity towards the fructo-furanosyl ring of sucrose (53
). Moreover, the Ile residue at position 2215 is also found in GTF-A from L. reuteri
) and is strongly conserved in the α-amylase family.
Likewise, in block E, the 2315
KGVQEKV peptide from CD2, following the second Asp of the catalytic triad, differs from the consensus sequence SEVQTVI found in most GTFs and DSRs. Besides, Arguello-Morales et al. (3
) also observed in the ASR sequence a specific tripeptide located at the same position, and it can also be noticed that GTF-A from L. reuteri
exhibits an original tripeptide just downstream of the third carboxylic acid of the triad (59
). In this conserved area, Glu2327 can be found in a position usually involved in the glucan structure determination. Indeed, the corresponding mutant T667R in DSR-S was found to synthesize a glucan with 13% α-1,3 linkages compared to less than 5% for the wild-type enzyme (43
). Consistent with this result, it appears that the presence of a carboxylic acid instead of a neutral amino acid (threonine) at the corresponding position of GTF-S increased the synthesis of α-1,3 glucosidic bonds by 30% (49
). In addition, concerning GTF-I from S. downei
, Monchois et al. (34
) also attribute an influence of D569 on oligosaccharide synthesis trough interaction with the acceptor molecule. Thus, DSR-E possesses, because of its two catalytic domains, both a neutral residue in CD1, Thr643, and an acidic residue in CD2, Glu2327.
Moreover, when aligning the DSR-E catalytic domain sequences with other central regions of glucansucrases, stretches of sequence are significantly longer, for example, between the general acid catalyst and the second aspartic acid residue of the catalytic triad in CD2, or shorter, as shown by a 16-amino-acid gap located upstream of the Ca2+ binding site of CD2 (data not shown).
In addition to the attentive study of both catalytic domain sequences, the question is, as no glucansucrase with two catalytic domains has been previously observed, how does such a molecule work? DSR-E possesses two nucleophiles (D527 and D2210) and all the conserved residues required for the formation of two glucosyl enzyme complexes. Thus, DSR-E seems to possess two fully active catalytic domains, and we can assume that the specificity in synthesizing α-1,2 linkages is related to one of the two domains—most probably CD2, which presents a distinctive stretch of sequence compared to CD1. One domain (CD1) would catalyze the transfer of a glucose moiety from sucrose to a maltose residue to give panose, which in turn would be glucosylated, either in CD1, resulting in the addition of a new α-1,6 glucosidic bond, or in CD2, specific for the α-1,2 linkage. It can be suggested that the presence of the GBD enables CD1 and CD2 to be maintained in close proximity for an optimal branching of the polymer.
To determine whether both domains are active, deletion studies and site-directed mutagenesis must be performed to evaluate the influence of each catalytic domain on the activity and specificity of DSR-E.