Lactobacillus reuteri 121 uses the glucosyltransferase A (GTFA) enzyme to convert sucrose into large amounts of the ␣-D-glucan reuteran, an exopolysaccharide. Upstream of gtfA lies another putative glucansucrase gene, designated gtfB. Previously, we have shown that the purified recombinant GTFB protein/enzyme is inactive with sucrose. Various homologs of gtfB are present in other Lactobacillus strains, including the L. reuteri type strain, DSM 20016, the genome sequence of which is available. Here we report that GTFB is a novel ␣-glucanotransferase enzyme with disproportionating (cleaving ␣134 and synthesizing ␣136 and ␣134 glycosidic linkages) and ␣136 polymerizing types of activity on maltotetraose and larger maltooligosaccharide substrates (in short, it is a 4,6-␣-glucanotransferase). Characterization of the types of compounds synthesized from maltoheptaose by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), methylation analysis, and 1-dimensional 1 H nuclear magnetic resonance (NMR) spectroscopy revealed that only linear products were made and that with increasing degrees of polymerization (DP), more ␣136 glycosidic linkages were introduced into the final products, ranging from 18% in the incubation mixture to 33% in an enriched fraction. In view of its primary structure, GTFB clearly is a member of the glycoside hydrolase 70 (GH70) family, comprising enzymes with a permuted (␤/␣) 8 barrel that use sucrose to synthesize ␣-D-glucan polymers. The GTFB enzyme reaction and product specificities, however, are novel for the GH70 family, resembling those of the GH13 ␣-amylase type of enzymes in using maltooligosaccharides as substrates but differing in introducing a series of ␣136 glycosidic linkages into linear oligosaccharide products. We conclude that GTFB represents a novel evolutionary intermediate between the GH13 and GH70 enzyme families, and we speculate about its origin.Glucansucrase (GS) (or glucosyltransferase [GTF]) enzymes (EC 2.4.1.5) of lactic acid bacteria (LAB) use sucrose to synthesize a diversity of ␣-glucans with ␣136 (dextran; found mainly in Leuconostoc), ␣133 (mutan; found mainly in Streptococcus), alternating ␣133 and ␣136 (alternan; reported only in Leuconostoc mesenteroides), and ␣134 (reuteran; synthesized by GTFA and GTFO from Lactobacillus reuteri strains) glycosidic bonds (1,14,16,23,34).The first glycoside hydrolase 70 (GH70) family 3-dimensional (3D) structures, recently elucidated (9, 38), showed that the catalytic domains of GS enzymes possess a (␤/␣) 8 barrel structure similar to that of members of the GH13 family, confirming earlier secondary-structure predictions (4, 21). The core of the proteins belonging to the GH13 family comprises 8 ␤-sheets alternated with 8 ␣-helices. In GS enzymes, however, this (␤/␣) 8 barrel structure is circularly permuted (21). Also, the four conserved regions (regions I to IV) identified in members of the ␣-amylase family GH13 (31) are present in glucansucrases. However, as a consequence of the circular per...

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Aims: Broussonetia papyrifera (Saeh plant) has many qualities, the inner bark was the material for 'dluwang' papers on which Indonesian historical manuscripts were written, and the leaves have bioactive constituents of medicinal value, and antifungal activities. We investigated the diversity of yeast species associated with leaves from 6 months and 1.5 yearold plants, which is prerequisite to understand the phylloplane yeasts and plant interaction. Methodology and results:The yeasts were isolated from fresh leaves by washing and membrane filtration methods. A total of 16 leaf samples of 6 months and 1.5 year-old plants were collected from four locations in Java, Indonesia, and 2,543 yeast isolates were obtained. Based on similarity of colony morphology, 82 representative yeast isolates were selected and identified based on the sequence analyses of internal transcribed spacer (ITS) regions of rDNA. The identification result showed that they consisted of 17 genera and 32 species. Thirty six of representative yeast isolates belong to 11 genera (18 species) of the phylum Ascomycota and forty six isolates belong to 6 genera (14 species) of the phylum Basidiomycota. Phylogenetic trees showed that the yeast isolates are phylogenetically diverse and distributed in the phyla of Ascomycota (classes Saccharomycetales and Dothideomycetes) and Basidiomycota (classes Microbotryomycetes, Tremellomycetes, and Ustilaginomycetes). Conclusions, significance and impact of study: The phylloplane yeasts of B. papyrifera (Saeh plant) were taxonomically heterogeneous. This is the first report of the isolation and identification of phylloplane yeasts from B. papyrifera. Phylloplane yeasts may possess antagonistic activity to fungal plant pathogens in their natural habitats.