Dataset Information

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family.

ABSTRACT: A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50 degrees C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a beta-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.

SUBMITTER: Seo JM

PROVIDER: S-EPMC2042100 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family.

Seo Jeong Min JM Ji Geun Eog GE Cho Sang Hee SH Park Myung Soo MS Lee Hyong Joo HJ

Applied and environmental microbiology 20070629 17

A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50 degrees C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search ...[more]

PMID: 17601807

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Project description:Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited ?-l-arabinofuranosidase activity toward p-nitrophenyl-?-l-arabinofuranoside but no activity toward p-nitrophenyl-?-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of ?1,2- and ?1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the ?1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan.IMPORTANCE We identified two novel ?-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on ?1,2/3-l-arabinofuranosyl linkages, while the latter acts on the ?1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. The genes encoding these enzymes are located side by side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.

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Dataset Information

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family.

Publications

Characterization of a Bifidobacterium longum BORI dipeptidase belonging to the U34 family.

Similar Datasets

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