Abstract
Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. Previously, we cloned and characterized the hypBA2 gene as a β-L-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel β-L-arabinofuranosidase that liberates L-arabinose from the L-arabinofuranose (Araf)-β1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-338 is a critical residue for catalytic activity. This study provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the glycoside hydrolase family 127.
Highlights
Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants
This study provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the glycoside hydrolase family 127
Bacterial Strains and Culture Conditions—The Bifidobacterium strains grown in Gifu anaerobic medium (GAM) broth (Nissui) were as follows: B. longum JCM 1217 and JCM 7054; B. longum subsp. infantis JCM 1222; B. pseudolongum JCM 1205; B. adolescentis JCM 1275; B. breve JCM 1192, and B. bifidum JCM 1254
Summary
Materials—Extensin, potato lectin, Hyp-linked -L-arabinooligosaccharides, -Ara, and Araf-1,2-Araf-OMe (Ara2Me) were prepared as described previously (10). Dansylated Hyp-linked -L-arabinooligosaccharides were prepared as described by Gray (11). P-Nitrophenyl (pNP) substrates were obtained from Sigma. HypBA2-C⌬486 was expressed and purified as described previously (10). Expression and Purification of Recombinant HypBA1—The genomic DNA of B. longum JCM 1217 was extracted using a FastPure DNA kit (Takara) and used for PCR amplification of the gene for the BL0422 ortholog, hypBA1. The forward (5Ј-AAGGAGATATACATATGAACGTTACAATCACTTCCC-3Ј) and reverse (5Ј-TGCTCGAGTGCGGCCGCTCGACGCTGGAAGACA-3Ј) primers were designed from nucleotides 4 –22 and 1959 –1974, respectively, of BL0422 from B
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