The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes

Abstract

The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing ␣-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-␣-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave ␣1¡4 linkages, and synthesize linear ␣1¡6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of ␣1¡6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-␣-glucanotransferase activity with maltooligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB-and GtfCtype enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with ␣-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (␤/␣) 8 barrel. The GtfC 4,6-␣-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between ␣-amylase and glucansucrase enzymes.T he starch-and sucrose-acting enzymes of the glycoside hydrolase 13 (GH13) and GH70 families are evolutionarily related, displaying similar protein folds and activity mechanisms (retaining, covalent intermediate, double displacement mechanism, three catalytic residues), constituting clan GH-H (http://www .cazy.org) (B. Svensson and Š. Janeček, glycoside hydrolase family 13 in CAZypedia, available at http://www.cazypedia.org/, accessed 2 June 2015; M. Remaud-Simeon, glycoside hydrolase family 70 in CAZypedia, available at http://www.cazypedia.org/, accessed 2 June 2015). They differ in their overall activities, degrading or modifying ␣-glucan substrates (starch, maltodextrins, GH13) (1, 2) or synthesizing ␣-glucan products (from sucrose, GH70) (3), e.g., by the Lactobacillus reuteri 121 GtfA enzyme (4). GH13 proteins have three domains (A, B, and C) with a common catalytic (␤/␣) 8 fold (triosephosphate isomerase [TIM] barrel); their active site is located in an open cavity between the A and B domains (5, 6). GH70 proteins share this domain organization, but their (␤/ ␣) 8 barrel is circularly permuted. Moreover, they possess unique domains IV and V (7). Recently, we ha...

4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

“…In recent years several maltodextrins/starch converting enzymes have been identified within the GH70 family supporting the evolutionary relatedness of GH13 and GH70 families23242526. Firstly, it was found that L. reuteri 121 produced a GS-like enzyme that was inactive on sucrose.…”

“…Besides, GSs present a “U-fold” domain structure in which 4 (domains A, B, IV and V) of the 5 domains are built up from two discontinuous segments of the polypeptide chain122021. During their evolution from GH13, the GH70 enzymes appear to have undergone a sequence of gene rearrangements that resulted in this unusual, circularly permuted domain organization23.…”

“…Following the annotation of many new genome sequences, a few family GH70 enzymes also were found in non-LAB genera. Previously we have characterized the GtfC enzyme of Exiguobacterium sibiricum 255–1523 and the GtfD enzyme of Azotobacter chroococcum NCIMB 800326. Both of these enzymes are inactive with sucrose and active with maltodextrins/starch, displaying 4,6-α-GTase activity that resulted in synthesis of isomalto/malto oligosaccharides (IMMO) (GtfC)23 and in a reuteran type of α-glucan (GtfD)26.…”

“…Previously we have characterized the GtfC enzyme of Exiguobacterium sibiricum 255–1523 and the GtfD enzyme of Azotobacter chroococcum NCIMB 800326. Both of these enzymes are inactive with sucrose and active with maltodextrins/starch, displaying 4,6-α-GTase activity that resulted in synthesis of isomalto/malto oligosaccharides (IMMO) (GtfC)23 and in a reuteran type of α-glucan (GtfD)26. Surprisingly, the domain organization in GtfC and GtfD resembles that of GH13 enzymes, with a nonpermuted order of conserved regions I-II-III-IV, and lacking domain V found in other GH70 enzymes.…”

“…For GSs, the crystal structure of GTF180-ΔN with a bound maltose acceptor combined with site-directed mutagenesis experiments revealed that the linkage specificity is controlled by residues forming the acceptor substrate binding subsites +1/+22029303132. Despite their different domain organization, GtfB, GtfC, and GtfD 4,6-α-GTase enzymes display high conservation in their motifs I to IV, particularly in residues forming the acceptor substrate binding subsites in GSs2326. Interestingly, a large number of these residues conserved in these 4,6-α-GTases differ in the GSs group of enzymes allowing the 4,6-α-GTase activity to be predicted by the analysis of the signature motifs I to IV.…”