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BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the Exiguobacterium sibiricum 255-15 GtfC enzymes. Both enzymes catalyze the cleavage of (α1→4) linkages in maltodextrin/starch and the synthesis of consecutive (α1→6) linkages. Here we describe a novel GH70 enzyme from the nitrogen-fixing Gram-negative bacterium Azotobacter chroococcum, designated as GtfD.METHODS: The purified recombinant GtfD enzyme was biochemically characterized using the amylose-staining assay and its products were identified using profiling chromatographic techniques (TLC and HPAEC-PAD). Glucans produced by the GtfD enzyme were analyzed by HPSEC-MALLS-RI, methylation analysis, 1D/2D Lombard et al. (2014) H/ Machius et al. (1995) C NMR spectroscopy and enzymatic degradation studies.RESULTS: The A. chroococcum GtfD is closely related to GtfC enzymes, sharing the same non-permuted domain organization also found in GH13 enzymes and displaying 4,6-α-glucanotransferase activity. However, the GtfD enzyme is unable to synthesize consecutive (α1→6) glucosidic bonds. Instead, it forms a high molecular mass α-glucan with alternating (α1→4) and (α1→6) linkages from amylose/starch, highly similar to the reuteran polymer synthesized by the L. reuteri GtfA glucansucrase from sucrose.CONCLUSIONS: In view of its origin and specificity, the GtfD enzyme represents a unique evolutionary intermediate between family GH13 (α-amylase) and GH70 (glucansucrase) enzymes.GENERAL SIGNIFICANCE: This study expands the natural repertoire of starch-converting enzymes providing the first characterization of an enzyme that converts starch into a reuteran-like α-glucan polymer, regarded as a health promoting food ingredient.

Nine GtfB-like 4,6-α-glucanotransferases (4,6-α-GTs) (represented by GtfX of L. aviarius subsp. aviarius DSM 20655) were identified to show distinct characteristics in conserved motifs I-IV. In particular, the "fingerprint" Tyr in motif III of these nine GtfB-type 4,6-α-GTs was found to be replaced by a Trp. In L. aviarius subsp. aviarius DSM20655, a second GtfB-like protein (GtfY), containing the canonical GtfB Tyr residue in motif III, was located directly upstream of GtfX. Biochemical characterization revealed that both GtfX and GtfY showed GtfB-like 4,6-α-GT activity, cleaving (α1→4) linkages and catalyzing the synthesis of (α1→6) linkages. Nonetheless, they differ in product specificity; GtfY only synthesizes consecutive (α1→6) linkages, yielding linear α-glucan products, but GtfX catalyzes the synthesis of (α1→6) linkages predominantly at branch points (22%) rather than in linear segments (10%). The highly branched α-glucan produced by GtfX from amylose V is resistant to digestion by α-amylase, offering great potential as dietary fibers.