The Exiguobacterium sibiricum 255-15 GtfC Enzyme Represents aNovel Glycoside Hydrolase 70 Subfamily of 4,6-�-GlucanotransferaseEnzymes

Joana Gangoiti,a Tjaard Pijning,b Lubbert Dijkhuizena

Microbial Physiologya and Biophysical Chemistry,b Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, TheNetherlands

The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacte-ria synthesizing �-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lac-tobacillus enzymes as 4,6-�-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of en-zymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave �1¡4 linkages, andsynthesize linear �1¡6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosac-charides into isomalto/malto-polysaccharides with a relatively high percentage of �1¡6 linkages. This paper reports the identi-fication of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacteriumsibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-�-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequenceidentity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with �-amylase domains A, B, andC 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 representstructurally and functionally very interesting evolutionary intermediates between �-amylase and glucansucrase enzymes.

The starch- and sucrose-acting enzymes of the glycoside hydro-lase 13 (GH13) and GH70 families are evolutionarily related,

displaying similar protein folds and activity mechanisms (retain-ing, covalent intermediate, double displacement mechanism,three catalytic residues), constituting clan GH-H (http://www.cazy.org) (B. Svensson and Š. Janecek, glycoside hydrolase family13 in CAZypedia, available at http://www.cazypedia.org/, accessed2 June 2015; M. Remaud-Simeon, glycoside hydrolase family 70 inCAZypedia, available at http://www.cazypedia.org/, accessed 2June 2015). They differ in their overall activities, degrading ormodifying �-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 pro-teins have three domains (A, B, and C) with a common catalytic(�/�)8 fold (triosephosphate isomerase [TIM] barrel); their activesite 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 uniquedomains IV and V (7). Recently, we have shown that truncation ofdomain V of glucansucrase Gtf180 of Lactobacillus reuteri 180heavily impairs its polysaccharide-synthesizing ability (8). Thefunction of domain IV in glucansucrase enzymes remains un-known.

The catalytic domains of GH13 enzymes display four con-served regions (I to IV) containing the catalytic residues and ac-tive-site residues involved in substrate and/or product binding.Interestingly, the catalytic domains of GH70 enzymes contain thesame conserved sequences but in a different order (II-III-IV-I);during their evolution from GH13, the GH70 enzymes thus ap-pear to have undergone a circular permutation but maintain full

functionality (7, 9). It remains unclear when GH13 enzymesevolved this permutation and/or GH70 enzymes gained this per-mutation, most of all because of a complete lack of GH13-GH70evolutionary intermediates.

Recently, we have reported the identification of a novel GH70subfamily, the GtfB-type enzymes that are unable to use sucrosefor �-glucan synthesis. Instead, GtfB enzymes act on starch/maltodextrin substrates catalyzing a 4,6-�-glucanotransferase re-action, cleaving �1¡4 linkages and introducing �1¡6 linkages inlinear product chains. This results in the synthesis of isomalto/malto-polysaccharides (IMMPs) that are digested not by humanenzymes but by the gut microbiota, thus acting as a soluble starchdietary fiber (10–14). GtfB enzymes have a domain organizationsimilar to that of glucansucrases but likely differ in the architec-ture of their active site (12).

Here we report the identification of a second GH70 subfamilyrepresented by the Exiguobacterium sibiricum 255-15 GtfC en-

Received 20 October 2015 Accepted 13 November 2015

Accepted manuscript posted online 20 November 2015

Citation Gangoiti J, Pijning T, Dijkhuizen L. 2016. The Exiguobacterium sibiricum255-15 GtfC enzyme represents a novel glycoside hydrolase 70 subfamily of 4,6-�-glucanotransferase enzymes. Appl Environ Microbiol 82:756 –766.doi:10.1128/AEM.03420-15.

Editor: C. Vieille

Address correspondence to Lubbert Dijkhuizen, L.Dijkhuizen@rug.nl.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03420-15.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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zyme. E. sibiricum 255-15 is a psychrotrophic bacterium isolatedfrom 3-million-year-old Siberian permafrost that grows from�5°C to 39°C (15). Evidence has been presented that this strainproduces both intracellular storage granules and capsular polysac-charides (15, 16). The physiological and functional roles of thisGtfC enzyme remain to be established. We show that the GtfC-and GtfB-type enzymes are biochemically related, both acting as4,6-�-glucanotransferase enzymes. Surprisingly, the domain or-der in GtfC resembles that of GH13 enzymes, with a nonpermutedorder of conserved regions I to IV and lacking the domain V foundin other GH70 enzymes. The new GtfC subfamily enzymes thusmay represent structurally very interesting evolutionary interme-diates between GH13 �-amylase and GH70 glucansucrase en-zymes, allowing further analysis of the evolutionary origins anddifferentiation of the (sub)families in clan GH-H (http://www.cazy.org).

MATERIALS AND METHODSPhylogenetic analysis. The E. sibiricum 255-15 sequence (GenBank ac-cession no. ACB62096.1) was retrieved from the GH70 database (http://www.cazy.org/) and used as the query in BLASTp searches (http://www.ncbi.nlm.nih.gov/BLAST/). Using the Pfam database (http://pfam.sanger.ac.uk/), the sequence was analyzed for the presence of a predicted GHdomain. Representative full-length GH70 and GH13 sequences identifiedvia BLASTp were used as the basis for the construction of a phylogenetictree. Sequences were aligned by MUSCLE in MEGA, version 6 (17), byusing default parameters. A phylogenetic analysis was performed inMEGA6 by the maximum-likelihood method based on the JTT matrix-based model. Partial deletion of the positions containing alignment gapsand missing data was conducted. Statistical confidence of the inferredphylogenetic relationships was assessed by performing 1,000 bootstrapreplicates. For the GenBank accession numbers of the sequences used inthis section, see Table S1 in the supplemental material.

Protein sequence analysis. The presence of a signal peptide in GtfCwas analyzed by using the Signal P4.1 server (http://www.cbs.dtu.dk/services/SignalP/). Searches for conserved domains were performed byusing the Pfam server. Pairwise sequence alignments between the func-tional regions of the E. sibiricum GtfC protein identified by the Pfamserver and the L. reuteri 121 GtfB protein were performed by using Jalview(18). The domain organization of GtfB was predicted by using the three-dimensional structures of L. reuteri 180 Gtf180-�N (encoding Gtf180amino acids 742 to 1772; Protein Data Bank [PDB] code 3KLK) (7) and L.reuteri 121 GtfA-�N glucansucrase (encoding GtfA amino acids 741 to1781; PDB code 4AMC) (19). Only the polypeptide segments correspond-ing to domains A, B, C, and IV were taken into account (Gtf180-�Nresidues 795 to 1638, GtfA-�N residues 794 to 163, and GtfB residues 762to 1619). Sequence alignments were generated with the ClustalW2 pro-gram (http://www.ebi.ac.uk/Tools/msa/clustalw2).

Cloning of the gtfC gene. Appropriate primer pairs (Table 1) wereused to create four different expression constructs with N-terminal Histags, one for the full-length GtfC protein without its putative signal pep-tide-encoding sequence (amino acids 31 to 893) and three for differentC-terminally truncated variants (amino acids 31 to 730, 31 to 743, and 31to 761).

The full-length gtfC gene was amplified by PCR from E. sibiricum255-15 (DSM 17290) chromosomal DNA with Phusion DNA polymerase(Finnzymes, Helsinki, Finland) and cloned into a modified pET15b vectorby ligation-independent cloning (LIC). Essentially, the gtfC gene was am-plified by PCR with primers with 5= extensions that facilitated the LICprocedure (forward and reverse). The KpnI-digested vector and the gtfCPCR product were then treated with T4 DNA polymerase (New EnglandBioLabs) in the presence of dTTP and dATP, respectively. The two reac-tion products were mixed together in a 1:4 molar ratio, and the mixturewas used to transform Escherichia coli DH5� cells (Phabagen, Utrecht,

The Netherlands). This resulted in a gtfC construct containing an N-ter-minal His6 tag cleavable by a 3C protease. The same procedure was used toconstruct the different C-terminally truncated variants. The pET15b/gtfC-based expression vectors constructed were transformed into the hostE. coli BL21 Star (DE3). The gene sequences were verified by nucleotidesequencing (LGC Genomics, Berlin, Germany).

Protein expression and purification. Overnight cultures of E. coliBL21 Star (DE3) harboring the gtfC gene were diluted 1/100 in Luria brothsupplemented with ampicillin (100 �g ml�1) and glucose (1%, wt/vol)and cultivated at 37°C with shaking at 240 rpm. When the culturesreached an optical density at 600 nm of �0.6, the inducer isopropyl-�-D-thiogalactopyranoside was added to a final concentration of 0.1 mM andcultivation was continued for 16 h at 16°C. The cells were then harvestedby centrifugation (10,000 � g, 20 min) and subsequently disrupted withB-PER lysis reagent in accordance with the manufacturer’s instructions(Pierce, Rockford, IL, US). After centrifugation (15,000 � g, 20 min), theclear supernatants were subjected to Ni–immobilized-metal affinity chro-matography. After being washed with 10 mM imidazole in 20 mM Tris-HCl (pH 8.0) and 1 mM CaCl2, the proteins were eluted with 200 mMimidazole in the same buffer. Further purification was performed by an-ion-exchange chromatography on a HiTrap column (1 ml; GE Health-care) using a linear gradient of elution of NaCl (0 to 1 M) in 20 mMTris-HCl buffer (pH 8.0) and 1 mM CaCl2 at a flow rate of 1 ml/min.Fractions of 1 ml were collected with an ÄKTA fast protein liquid chro-matography system (GE Healthcare, Uppsala, Sweden). The GtfC-con-taining fractions were concentrated and exchanged into 20 mM Tris-HClbuffer–1 mM CaCl2 in a stirred ultrafiltration unit (Amicon, Beverly, MA)on a membrane with a 30,000 molecular weight cutoff. Purity and homo-geneity were analyzed by SDS-PAGE, and protein concentrations weredetermined with a NanoDrop 2000 spectrophotometer (Isogen Life Sci-ence, De Meern, The Netherlands).

Enzyme assays. The initial total GtfC enzyme activity was determinedby the amylose-iodine assay described by Bai et al. (20), with minor mod-ifications. This method is based on monitoring of the decrease in theabsorbance at 660 nm of the �-glucan-iodine complex resulting fromtransglycosylation and/or hydrolytic activity (20). The reaction mixturecontained 0.125% (wt/vol) amylose V (AVEBE, Foxhol, The Netherlands)in 25 mM sodium acetate buffer (pH 6.0) in a total volume of 325 �l. Afterincubation at 40°C for 10 min, the reaction was started by the addition of8 �g of the purified enzyme. Every 2 min for a total of 16 min, samples of25 �l were taken and 12.5 �l of 0.4 M NaOH was added to stop thereaction. Subsequently, the reaction mixture was neutralized by the addi-tion of 12.5 �l of 0.4 M HCl. These samples were used to quantify theamylose content by the iodine staining method (20). One unit of activitycorresponds to the amount of enzyme converting 1 mg of amylose V/min.The optimal pH and temperature were determined over a pH range of 3.5to 9.5 and a temperature range of 30 to 55°C. Sodium acetate buffer (25mM) was used at pH 3.5 to 6.0, morpholinepropanesulfonic acid (MOPS)buffer (25 mM) was used at pH 6.0 to 7.0, sodium phosphate buffer (25mM) was used at pH 6.0 to 7.5, Tris-HCl buffer (25 mM) was used at pH7.0 to 8.0, and glycine-NaOH buffer (25 mM) was used at pH 9.0 to 9.5.For thermostability studies, the enzyme (0.5 mg/ml) was incubated in theabsence of a substrate for 10 min at different temperatures ranging from50 to 95°C and then immediately cooled to 4°C. The residual activity wasmeasured as described above.

Substrate/product analysis of GtfC. Purified GtfC enzyme (40 �g/ml) was incubated separately with 25 mM sucrose (Acros), nigerose (Sig-ma-Aldrich), panose (Sigma-Aldrich), isomaltose (Sigma-Aldrich),isomaltotriose (Sigma-Aldrich), isomaltopentaose (Carbosynth), malto-oligosaccharides (MOS) with different degrees of polymerization (G2 toG7) (Sigma-Aldrich), and 0.6% (wt/vol) amylose V (AVEBE, Foxhol, TheNetherlands). All reactions were performed in 25 mM sodium acetatebuffer, pH 6.0, with 1 mM CaCl2 at 37°C for 24 h. The reactions werestopped by incubation at 90°C for 10 min. The progress of the reactions

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was monitored by high-performance anion-exchange chromatography(HPAEC) (described below) and/or thin-layer chromatography (TLC).

TLC and HPAEC with pulsed amperometric detection (HPAEC-PAD) analysis. Samples were spotted in 1-cm lines on TLC sheets (MerckSilica Gel 60 F254, 20 by 20 cm) and separated by a solvent system ofn-butanol–acetic acid–water (2:1:1, vol/vol). The bands were visualizedby orcinol-sulfuric acid staining and compared with a simultaneous runof a mixture of glucose and MOS (degree of polymerization of 2 [DP2] toDP7).

Product mixtures from incubations with GtfC were analyzed byHPAEC on a Dionex DX500 work station (Dionex, Amsterdam, TheNetherlands) equipped with a CarboPac PA-1 column (Dionex; 250 by 4mm) and an ED40 pulsed amperometric detector. The oligosaccharideswere separated at a 1-ml/min flow rate by using a linear gradient of 10 to240 mM sodium acetate in 100 mM NaOH. The identities of the peakswere assigned by using commercial oligosaccharide standards.

NMR spectroscopy. One-dimensional 1H nuclear magnetic reso-nance (NMR) spectra of the product mixtures were recorded in D2O on aVarian Inova 500 spectrometer (NMR Center, University of Groningen)at a probe temperature of 300 K and processed with MestReNova 5.3(Mestrelabs Research SL, Santiago de Compostella, Spain). Prior to anal-ysis, samples were exchanged twice in D2O (99.9 atom% D; CambridgeIsotope Laboratories, Inc., Andover, MA) with intermediate lyophiliza-tion and then dissolved in 0.6 ml of D2O containing acetone ( 2.225ppm) as an internal standard.

RESULTS AND DISCUSSIONIdentification of a novel GH70 subfamily. Recently, the Carbo-hydrate-Active enZYmes (CAZy) database (http://www.cazy.org)reported the presence of two new and almost identical GH70 pro-teins in E. sibiricum 255-15 (GenBank accession no. ACB62096.1)and Exiguobacterium antarcticum B7 (GenBank accession no.AFS71545.1) (100% coverage, 93% identity) annotated as dex-transucrases. Thus far, all GH70 family members have been foundin lactic acid bacteria (in the genera Leuconostoc, Streptococcus,Lactobacillus, and Weissella) (21). Exiguobacterium also is a genuswithin the class Bacilli and a member of the low-GC phylum Fir-micutes but does not belong to the lactic acid bacteria. E. sibiricum255-15 and E. antarcticum B7 are free-living, psychrotrophic,nonsporulating, Gram-positive bacteria. E. sibiricum is prevalentin the Siberian permafrost (15, 16). E. antarcticum B7 has beenisolated from a microbial biofilm at Ginger Lake on King GeorgeIsland, Antarctic Peninsula (22). We decided to carry out a moredetailed bioinformatic analysis of the GH70 enzyme from E. sibiri-cum 255-15, designated GtfC.

A BLASTp search with GtfC protein as the query sequenceidentified nine additional homologs of this enzyme present in theExiguobacterium and Bacillus genera, constituting a new GH70subfamily (see Table S2 in the supplemental material). The E.sibiricum 255-15 GtfC protein sequence is 75% identical to pro-

teins annotated as dextransucrases encoded in the genomes ofExiguobacterium undae, E. antarcticum (strain DSM 14480), E.sibiricum (strain 7-3), E. acetylicum (strain DSM 20416), and Ex-iguobacterium sp. strain RIT341. Also, in the recently elucidatedgenome sequences of various Bacillus strains (Bacillus kribbensisDSM 17871, Bacillus coagulans DSM 1, and Bacillus coagulans2-6), we identified homologs of GtfC that are 55% identical to it.The sequences of (putative) GtfB-like 4,6-�-glucanotransferaseswere retrieved as the next hits of the BLASTp analysis with statis-tically significant E values (ranging from 6e�60 to 4e�49) butwith amino acid sequences only 29 to 35% identical to that of E.sibiricum 255-15 GtfC. The E. sibiricum 255-15 GtfC homologspresent in Exiguobacterium and Bacillus strains form a separatebranch closely related to GtfB-like 4,6-�-glucanotransferases andclearly positioned between the GH70 and GH13 family proteins(Fig. 1). In a previous study, we reported that GtfB-like proteinsform a small cluster clearly separate from the glucansucrase-typeenzymes, with just six members (12). Because of the increasednumber of genome sequences available as of June 2015, theGtfB-like 4,6-�-glucanotransferase GH70 subfamily contains22 proteins. All of these GtfB homologs are encoded by Lacto-bacillus strains, with the exception of the hypothetical GH70 pro-tein of Pediococcus pentosaceus IE-3. The last hits obtained byBLAST were (putative) glucansucrases present in the genomesof lactic acid bacteria of the genera Streptococcus, Lactobacillus,Leuconostoc, Oenococcus, and Weissella, followed by GH13 pro-teins, including well-recognized enzymes such as the �-amylasefrom B. stearothermophilus (coverage, 57%; identity, 25%; E value,4e�12) (Fig. 1).

Domain organization of E. sibiricum GtfC and GtfC-like en-zymes. GtfC of E. sibiricum 255-15 was chosen as a representativeto analyze the domain organization of the novel GH70 GtfC sub-family. The complete nucleotide sequence for this enzyme en-codes a polypeptide of 893 amino acids with a calculated molecu-lar mass of 99 kDa. An N-terminal conserved Gram-positivesignal sequence of 30 amino acids was predicted by the signalsequence predictor Signal P4.1, suggesting that GtfC functions asan extracellular enzyme. The E. sibiricum 255-15 GtfC proteinsequence was examined with the Pfam sequence search server,resulting in the annotation of different segments (Fig. 2). First,two segments of the sequence (residues 15 to 200 [GH70n] andresidues 314 to 761 [GH70c]) were associated with the GH70 fam-ily. Second, in the C-terminal part of the sequence, a segmentcovering residues 730 to 888 was identified as a pair of bacterialimmunoglobulin-like domains (group 2) of �75 amino acid res-idues each (Ig2). Although this Ig2 segment partially overlaps theGH70c segment, the alignment confidence suggests that the over-

TABLE 1 Primers used in this study

Primer Application Sequence (5=¡3=)a

Fw31Esib Cloning in expression vector forward primer CAGGGACCCGGTTATACGTCAGGTGAGAAATTGRv893Esib Cloning in expression vector reverse primer CGAGGAGAAGCCCGGTTACTTCACGATGACTTTGAAGGRv730Esib Cloning in expression vector reverse primer CGAGGAGAAGCCCGGTTATGCTTTTGTTGTTTGTGGAACRv743Esib Cloning in expression vector reverse primer CGAGGAGAAGCCCGGTTATCCTTGATAGACACTTGCTTTCRv761Esib Cloning in expression vector reverse primer CGAGGAGAAGCCCGGTTACGATGATACAGTTGATGACGTATTCSeqES1 Nucleotide sequencing CAGGATTTCCGCTTCAAGCCSeqES2 Nucleotide sequencing AGTGTTCTTTCGCGACTTGGa LIC 5= overhangs are in bold.

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lapping residues (730 to 761) most likely belong to an Ig2 domain.Sequence analysis of putative GtfC-like homologues suggests thatIg2-like domains are not always present. Ig2-like domains arefound in a variety of bacterial and phage surface proteins; e.g., in E.coli intimins, they mediate cell surface adhesion (23, 24). Anotherpossibility is that they function as carbohydrate-binding modules(25, 26).

Surprisingly, although the E. sibiricum 255-15 GtfC proteinand its homologs showed the highest sequence identity with GH704,6-�-glucanotransferases, a pairwise alignment of GtfC withGtfB (Fig. 2) revealed that the two annotated GH70 segments ofGtfC are conversely ordered. Indeed, the N-terminally locatedGH70n segment of GtfC aligned with the predicted C-terminal

segments of domains A and B of GtfB, while the C-terminallylocated GH70c segment aligned with the N-terminal parts of do-mains B and A and with domain C of GtfB. The segment locatedbetween the two GH70 segments shows homology to domain IVof GtfB, again with two half segments (residues 201 to 257 and 258to 313) ordered conversely. Combining the aligned segments al-lowed us to propose a domain organization for E. sibiricum 255-15GtfC (and its homologs) (Fig. 3) in which the polypeptide chainfrom the N to the C terminus successively contributes to the An,Bn, IV, Bc, Ac, and C domains and, starting from residue 730, thetwo Ig2-like domains. Thus, the domain organization of the coredomains (A, B, and C) of GtfC-like proteins unexpectedly resem-bles that of GH13 �-amylases, lacking the circular permutation of

FIG 1 Phylogenetic tree of representative GH70 and GH13 protein sequences identified via BLASTp searches with the E. sibiricum 255-15 GtfC 4,6-�-glucanotransferase protein as the query. The evolutionary history was inferred by the maximum-likelihood method based on the JTT matrix-based model. Thebar corresponds to a genetic distance of 0.2 substitution per position (20% amino acid sequence difference). The bootstrap values adjacent to the main nodesrepresent the probabilities based on 1,000 replicates. The protein sequences are annotated by their GenBank GenInfo Identifier sequence identification numbers.For the names of the bacterial species, see Table S1 in the supplemental material. The novel subfamily of GtfC-like 4,6-�-glucanotransferase enzymes is on a graybackground.

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the (�/�)8 barrel that is observed in GH70 glucansucrases. In ad-dition to the core domains, domain IV (of unknown function, sofar found only in GH70 glucansucrases) is present as an insertionin domain B and several GtfC-like enzymes contain one or twoIg2-like domains. On the other hand, both the variable N-termi-nal domain and domain V, typical for glucansucrases (21), seemto be absent from GtfC-like enzymes.

Reaction specificity of the GtfC subfamily. The finding ofGH70 sequences in a mostly GH13-like domain organizationprompted us to analyze the four homology regions (I to IV) con-served in the members of the GH13 and GH70 families (http://www.cazy.org) in more detail for the GtfC subfamily, as theseregions that contain the catalytic and substrate-binding residuesgive clues to their reaction specificity (27). Indeed, regions I to IVwere easily identified in a sequence alignment of enzymes of bothGH families (Fig. 4), revealing high conservation. First, the sevenamino acid residues that are conserved in motifs I to IV of mostfamily GH13 members (5) are fully conserved in the GH70 family(27), except His140 (BSTA B. stearothermophilus �-amylase num-bering), which is replaced by Gln in all GH70 enzymes (Gln1484,

GtfB L. reuteri 121 numbering). Similar to GH70 proteins, theequivalent residue in the GtfC-like group of enzymes also is a Gln.Among the seven residues, the three (putative) catalytic residueswere also identified in all members of the GtfC subfamily of 4,6-�-glucanotransferases (D407, E438, and D509 [E. sibiricum GtfCnumbering]). Otherwise, a large number of amino acid residuesconserved in regions I, II, III, and IV in the GtfB-like 4,6-�-glu-canotransferase enzymes are conserved in E. sibiricum GtfC andhomologs. In GtfC homologs, the conserved residue W1065(Gtf180 L. reuteri 180 numbering) is replaced by a Tyr, as is thecase in GtfB-like 4,6-�-glucanotransferases. Moreover, the aminoacid residues at positions 1137, 1140, and 1141, known to be im-portant for glucosidic linkage specificity in glucansucrases (28–30), are Gln, Lys, and Asn, respectively, in GtfC homologs, similarto GtfB-like 4,6-�-glucanotransferases. Apart from the well-rec-ognized I-to-IV homology regions, the three additional conservedregions (VI, V, and VII) proposed to be related to the maintenanceof the structure and to a given enzyme specificity in GH13 familyproteins (6, 31, 32) were identified in the GH70 and GH13 se-quences. Interestingly, GtfC- and GtfB-like proteins had some

FIG 2 Circular permutation of domains in GtfB and GtfC 4,6-�-glucanotransferase enzymes. Sequence numbers represent the start of each predicted domain.Left, predicted GtfB domain organization plotted along the primary structure (20). Right, primary structure of the E. sibiricum GtfC protein showing functionalregions identified by the Pfam server, annotated with information about the pairwise alignment of the different regions, the predicted domain order, and thelocations of conserved regions I to IV in GtfC. aa, amino acids.

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specific features in common in these motifs. Both enzyme typesdisplayed high conservation in their homology region VI, the pep-tide flanked by the invariant Gly and Pro residues, and possessedThr, Asp, and Trp residues at positions 73, 74, and 76, respectively(E. sibiricum GtfC numbering). Besides, GtfC and GtfB homo-logues have an invariant Thr residue at the beginning of motif VII,instead of the well-conserved Gly residue typically found in GH13family members. It should be pointed out that the consensus cal-cium-binding Asp residue present in region V of most GH13 pro-teins was also strictly conserved within the GH70 family, indicat-ing that GtfC enzymes, like other GH70 and GH13 proteins,require calcium ions (7, 31). In conclusion, regarding their aminoacid sequences, the GtfC-like enzymes are 4,6-�-glucanotrans-ferase-type enzymes belonging to GH70.

Evolutionary events leading to the circularly permuted orga-nization of GH70 members from a GtfC enzyme intermediate.As shown above, the domain organization of GtfC resembles thatof GH13 enzymes. Consequently, and in contrast to the permutedorder II-III-IV-I characteristic of GH70 glucansucrases and GtfBhomologues, the sequence order of the four homology regions inGtfC homologs is nonpermuted (I-II-III-IV) (Fig. 3). This clearlysuggests that the GtfC-type enzymes represent an evolutionary inter-mediate subfamily between the GH13 and GH70 enzymes (Fig. 3)with a domain organization resembling that of GH13 enzymes andenzymatic reaction specificities resembling those of GH70 enzymes,especially GtfB 4,6-�-glucanotransferases (see below).

Earlier, Vujicic-Žagar et al. proposed an evolutionary pathwayleading to the unusual, circularly permuted domain organizationof GH70 glucansucrases from a putative GH13 ancestor �-amy-lase precursor (7). In this pathway, it was speculated that insertionof domain IV may have occurred either before or after the events(gene duplication and partial truncation) that lead to circular per-mutation. With the discovery of the GtfC subfamily presentedhere, only the first scenario appears to be possible: insertion ofdomain IV in domain B of the ancestor �-amylase leads to a (non-permuted) domain organization observed in GtfC-like enzymes,of which some but not all acquired Ig-like domains at the C ter-minus. The other “branch” would then continue with the eventsleading to the circularly permutated domain organization withone more inserted domain (V) observed in glucansucrases andGtfB-like enzymes (Fig. 5).

Purification and biochemical properties of the E. sibiricum255-15 GtfC enzyme. Full-length GtfC (amino acids 31 to 893)was cloned and expressed in E. coli. Under the conditions used,relatively low expression levels were observed in the soluble andinsoluble fractions (see Fig. S1 in the supplemental material). Re-combinant GtfC was purified from the soluble fraction by His tagaffinity and anion-exchange chromatography yielding �0.35 mgof pure protein per liter of E. coli culture. Aiming to improveprotein expression and to study the role of the Ig-like 2 domains,different C-terminally truncated variants were constructed (withamino acids 31 to 730, 31 to 743, and 31 to 761). However, in all

FIG 3 Schematic representations of the domain orders in GH13 �-amylases and GH70 glucansucrases (GSs) and 4,6-�-glucanotransferases (4,6-�-GTs).Regarding their domain organization, the GtfC-like enzymes likely represent an evolutionary intermediate between the GH13 �-amylases (left) and the GH70glucansucrases and GtfB-like 4,6-�-glucanotransferases (right). The U-shaped course of the polypeptide chain of GH70 glucansucrases was first identified in thecrystal structure of Gtf180-�N (7) (right) and results in four of the five domains being formed from N- and C-terminal halves. Domains A, B, C, IV, and V areblue, green, magenta, yellow, and red, respectively. Ig2-like domains are gray. As apparent from the order of the conserved regions, only the GH70 glucansucrasesand GH70 GtfB-like 4,6-�-glucanotransferases are circularly permuted (order: II-III-IV-I). The parentheses indicate that the Ig2-like domains are not identifiedin all GtfC-like enzymes and that the C-terminal half of domain V is not found in GtfB-like 4,6-�-glucanotransferases.

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cases, these truncated proteins were insoluble and accumulated ininclusion bodies in E. coli.

Determination of the total activity of GtfC on amylose, in theabsence or presence of 1 mM CaCl2, revealed that the Ca2� ionsslightly activated GtfC enzyme activity, as is the case for L. reuteri121 GtfB (20). The effects of pH and temperature on the activity ofthe full-length GtfC enzyme were examined with amylose V as asubstrate. The enzyme exhibited its maximum activity in 25 mM

sodium acetate buffer, pH 6.0, containing 1 mM CaCl2 and re-tained more than 50% of this activity over a pH range of 4.0 to 7.5.GtfC was most active between 35 and 50°C, showing its maximalactivity at 45°C; its activity decreased drastically when the reactionwas carried out at 55°C. Despite being produced by a cold-adaptedbacterium, GtfC exhibited only 30% of its maximum activitywhen the temperature was decreased to 30°C. This GtfC enzyme isresistant to thermal inactivation up to 45°C in sodium acetate

FIG 4 Alignment of conserved sequence motifs I, II, III, IV, V, VI, and VII in the catalytic domains of (putative) GtfC-like 4,6-�-glucanotransferases (A),(putative) GtfB-like 4,6-�-glucanotransferases (B), glucansucrases (C), and GH13 �-amylases (D). The seven strictly conserved amino acid residues in motifs Ito IV of GH70 enzymes (numbered 1 to 7 above the sequences) are also conserved in the novel GH70 GtfC subfamily. Amino acids that constitute the catalytictriad are boxed. Residues forming acceptor-binding subsites �1, �1, and � 2 in Gtf180-�N are indicated by the numbers below the sequences. Gray shadingindicates amino acid conservation between residues according to BLOSUM62 scores. Abbreviations at the bottom: NU, nucleophile; A/B, general acid/base; TS,transition state stabilizer.

FIG 5 Hypothetical evolutionary pathway based on the “permutation per duplication model” (34) leading from a putative ancestor GH13 �-amylase precursorto GH70 GtfC-like 4,6-�-glucanotransferases and to GH70 glucansucrases (GSs) and GtfB-like 4,6-�-glucanotransferases (GTs). Insertion of domain IV leads tothe “intermediate” GtfC subfamily. Some, but not all, of the GtfC-like enzymes acquire Ig-like domains (route indicated by dotted arrows). Others undergo geneduplication, partial terminal deletions, and domain insertion events that lead to GH70 glucansucrases and GtfB 4,6-�-glucanotransferases. Sequence segmentsforming domains A, B, C, IV, and V are blue, green, magenta, yellow, and red, respectively. Ig2-like domains are gray.

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buffer, pH 6.0. The specific total activity value in 25 mM sodiumacetate buffer, pH 6.0, containing 1 mM CaCl2 at 40°C was 2.2 �0.1 U/mg, similar to that of GtfB-�N (under its optimal condi-tions of pH 5.0 and 40°C), namely, 2.8 U/mg (20).

Substrate and product specificity of the E. sibiricum 255-15GtfC enzyme. In order to confirm the predicted reaction specific-ity of GtfC-like enzymes, we examined the substrate specificity ofGtfC of E. sibiricum 255-15 by incubating the enzyme with differ-

FIG 6 TLC analysis of the products synthesized by the E. sibiricum 255-15 GtfC (A) and L. reuteri 121 GtfB (B) 4,6-�-glucanotransferase enzymes at 40 �g ml�1

from MOS (DP2 to DP7) and amylose V. The reaction mixtures were incubated at 37°C and pH 6.0 (GtfC) or pH 4.7 (GtfB) for 24 h. Std, standard; G1, glucose;G2, maltose; G3, maltotriose; G4, maltotetraose; G5, maltopentaose; G6, maltohexaose; G7, maltoheptaose; Amy, amylose V; Pol, polymer.

FIG 7 1H NMR analysis of product mixtures obtained from malto-oligosaccharides (DP4 to DP7) and amylose V (Amy) incubations with the E. sibiricum 255-15GtfC and L. reuteri 121 GtfB 4,6-�-glucanotransferase enzymes at 40 �g ml�1 for 24 h at 37°C and pH 6.0 (GtfC) or 4.7 (GtfB). (A) The 500-MHz 1H NMRspectrum of the oligosaccharide mixture generated after the incubation of maltoheptaose (DP7) with GtfC 4,6-�-glucanotransferase recorded in D2O at 25°C.Chemical shifts are shown in parts per million relative to the signal of internal acetone ( 2.225). The bar diagrams show the percentages of glucose and �1¡4and �1¡6 glycosidic linkages in the products synthesized by GtfC (B) and GtfB (C) enzymes.

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ent oligosaccharides and comparing its activity with that of L.reuteri 121 4,6-�-glucanotransferase GtfB. Indeed, incubation ofGtfC with MOS with DP4 to DP7 revealed the formation of arange of shorter and longer oligosaccharide products, reflecting itsmain disproportionation (transglycosidase) activity (Fig. 6A).This activity is similar to that of GtfB, which displayed clear hy-drolase/transglycosylase activity on MOS with DP4 to DP7 andaccumulated polymeric material, as well as different oligosaccha-rides (Fig. 6B). However, GtfC did not accumulate polymeric ma-terial from MOS. When amylose V was used as a substrate, GtfCproduced significantly more oligosaccharide products than GtfB,which produced mainly a (modified) polymer (IMMP) (Fig. 6Aand B) (13). The GtfC enzyme is unable to use maltose or malto-triose as a substrate, while GtfB showed low disproportionating

activity with maltotriose. Similar to the GtfB enzyme, GtfC wasinactive on sucrose; nigerose; iso-MOS with DP2, DP3, or DP5;panose; and reuteran (produced by the L. reuteri 121 GtfA en-zyme) (data not shown).

To analyze the MOS-specific transglycosylase reaction speci-ficity of GtfC in more detail, the different product mixtures wereanalyzed by one-dimensional 1H NMR spectroscopy. As an exam-ple, the 1H NMR spectrum of the product mixture generated frommaltoheptaose is depicted in Fig. 7A. 1H NMR analysis revealedthe presence of the anomeric signals at �5.40 and 4.97 corre-sponding to the �1¡4 linkages and the newly synthesized �1¡6linkages. The signal typical for (1¡4,6)-�-D-Glcp-(1¡4)branches (H-1, �5.36) was not detected, demonstrating thatGtfC acted on MOS and amylose-V yielding linear gluco-oligom-

FIG 8 HPAEC-PAD profiles of the oligosaccharide mixtures formed upon the incubation of E. sibiricum 255-15 GtfC with maltoheptaose (A) and amylose-V(B) for 10 min, 1 h, and 3 h (pH 6.5, 37°C). The identities of peaks were assigned by using commercial oligosaccharide standards. G1, glucose; G2 to G7, maltoseto maltoheptaose; iso-G2, isomaltose; iso-G3, isomaltotriose; Pa, panose.

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ers also containing, besides �1¡4, �1¡6 glycosidic linkages. Thespectra also revealed the presence of signals corresponding to freeglucose units (G� H-1, 5.225; G� H-1, 4.637) and 4-substi-tuted reducing-end glucose residues (R� H-1, 5.225; R� H-1, 4.652). Small signals corresponding to 6-substituted reducing-end glucose residues (R� H-1, 5.241; R� H-1, 4.670) were alsodetected. These trace amounts of 6-substituted reducing-end glu-cose units are also found in the 1H NMR spectra of the productmixtures produced by the GtfB-type enzyme, indicating that these4,6-�-glucanotransferases use glucose as an acceptor sub-strate and elongate it with one �1¡6-linked glucose residue at thenonreducing site (12). In summary, the 1H-NMR spectra ofthe products generated by GtfC presented signals characteristic ofthe linear isomalto-/malto-oligomer products also synthesized byGtfB (11); thus, GtfC acts as a 4,6-�-glucanotransferase. The per-centages of glucose and �1¡4 and �1¡6 glycosidic linkages inthe product mixtures were determined for GtfB and GtfC reactionproducts (Fig. 7B and C). With both enzymes, we observed thatthe higher the DP of the substrate, the higher the percentages of�1¡6 glycosidic linkages introduced into the product. The totalpercentage of �1¡6 glycosidic linkages introduced was lower inthe case of GtfC.

Oligosaccharides initially formed from maltoheptaose andamylose V by the E. sibiricum 255-15 GtfC enzyme. To study themode of action of GtfC, the oligosaccharides initially formed bythe enzyme from maltoheptaose and amylose V were analyzed byHPAEC (Fig. 8). When G7 (slightly contaminated with G6 andG5) was used as a substrate, the first clear products of the reactionwere G1 (glucose), G3 (maltotriose), and G6 (maltohexaose). Be-sides, small peaks at retention times longer than that of the donorwere detected that did not fit with MOS retention times and thatprobably correspond to oligosaccharides containing �1¡6 link-ages. The release of G3 at the beginning of the reaction suggeststhat GtfC has an additional endo-�1¡4-glycosidase activity, be-ing able to cleave off not only the nonreducing glucose unit butalso a maltotetraosyl unit and transfer it to another glucan chain.Later, oligosaccharides with DPs higher than that of the startingdonor started to accumulate. Besides glucose and MOS with DP2to DP7, peaks corresponding to isomaltose, isomaltotriose, andpanose were identified, confirming the ability of GtfC to synthe-size �1¡6 linkages. Incubation of amylose V with GtfC yieldedG1 (glucose) and G2 (maltose) as the main first products of thereaction. In time, a wide range of oligosaccharides are synthesizedas a result of GtfC hydrolase/transglycosidase activity. The morepronounced accumulation of smaller oligosaccharide productsby GtfC is in agreement with the endo-�1¡4-glycosidase ac-tivity observed when using G7 as a substrate. However, it alsomay indicate that GtfC possesses a less processive mechanismof polymerization than GtfB, This less processive mechanism ofpolymerization may be related to the absence of domain V in theE. sibiricum GtfC enzyme. Recently, we have demonstrated thattruncation of domain V of glucansucrase Gtf180 yielded an en-zyme that is incapable of polymer synthesis and produces largeamounts of oligosaccharides instead (8). Thus, domain V was pro-posed to be important for providing binding zones for the �-glu-can chains, facilitating elongation in a processive manner.

Conclusions. This paper reports biochemical characteristics ofthe GtfC enzyme of E. sibiricum 255-15. The data show that thisGtfC enzyme and its homologs in other Exiguobacterium and Ba-cillus strains represent a second GH70 subfamily with 4,6-�-glu-

canotransferase activity with MOS and amylose/starch, as previ-ously found in the GtfB-type enzymes of lactobacilli. The GtfCenzymes provide the first example of family GH70 enzymes innon-lactic-acid bacteria. Biochemically, GtfC is more closely re-lated to the GH70 GtfB enzymes, but its domain organization ismore similar to that of GH13 enzymes, especially in view of theorder of the four conserved regions of clan GH-H. The GtfC andGtfB subfamilies thus provide very interesting evolutionary inter-mediates between families GH13 and GH70. The carbohydrateproducts of these different (sub)families have been suggested toprotect bacterial cells against harsh environmental conditions orallow adherence at surfaces, i.e., assist in biofilm formation (3, 27).Lactic acid bacteria employ glucansucrase enzymes (e.g., GtfA) tosynthesize �-glucans with various glycosidic linkages from su-crose or GtfB enzymes to convert starch into IMMPs (13). Thesebacterial polysaccharides are clearly more difficult to digest byhydrolytic enzymes of environmental competitors (33), thus pro-viding a competitive advantage. Also, the carbohydrate productsof the GtfC enzymes from non-lactic-acid bacteria may have suchroles, but a function in protecting E. sibiricum and E. antarcticumcells against the freezing temperatures of their habitats cannot beexcluded either.

ACKNOWLEDGMENTS

We thank Justyna Dobruchowska for assistance with NMR analysis andAlicia Lammerts van Bueren for providing the LIC-compatible pET15bvector.

FUNDING INFORMATIONThis study was financially supported by the University of Groningen andby the TKI Agri&Food program as coordinated by the CarbohydrateCompetence Center (CCC-ABC; www.cccresearch.nl).

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