Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum

Fujita, Kiyotaka; Takashi, Yukari; Obuchi, Eriko; Kitahara, Kuninori; Suganuma, Toshihiko

doi:10.1074/jbc.m113.528711

Cited by 53 publications

(60 citation statements)

References 34 publications

(31 reference statements)

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“…By using site-directed mutagenesis, Fujita et al (13) were able to demonstrate that the Glu338 residue of the ␤-arabinofuranosidase (HypBA1) from Bifidobacterium longum is critical for catalytic activity. By comparing the polypeptide sequences of CpAbf127A and CpAbf127B with the amino acid sequence of the ␤-arabinofuranosidase from Bifidobacterium longum, it was possible to identify the corresponding amino acid in CpAbf127B as Glu336.…”

Section: Resultsmentioning

confidence: 99%

“…ABF enzymes are members of GH families 3, 43, 51, 54, and 62, whereas ABN enzymes belong solely to GH family 43. Recently, a recombinant ␤-arabinofuranosidase from Bifidobacterium longum was characterized, which led to the definition of GH family 127 (13). The characterized protein was able to hydrolyze glycoprotein-derived ␤-arabinofuranose residues.…”

Section: Abstract Pectic Enzymes Thermophilesmentioning

confidence: 99%

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Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Wefers

Dong

Abdelhamid

et al. 2017

Appl Environ Microbiol

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The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 ␣-L-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 ␤-L-arabinopyranosidase (CpAbp27A), and two GH127 ␤-Larabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are ␣-L-arabinofuranosidases that exhibit diverse substrate specificities, cleaving ␣-1,2, ␣-1,3, and ␣-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved ␤-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and L-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus. IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus. The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as ␤-L-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

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Section: Resultsmentioning

confidence: 99%

Section: Abstract Pectic Enzymes Thermophilesmentioning

confidence: 99%

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Wefers

Dong

Abdelhamid

et al. 2017

Appl Environ Microbiol

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“…Fujita et al demonstrated that HypBA1 also catalyzes the transglycosylation to methanol (14). Using 1 H and 13 C NMR analyses, the transglycosylation product was found to be methyl-β-Araf, indicating that HypBA1 follows a reaction mechanism that retains the configuration of the anomeric carbon.…”

Section: E Proposed Reaction Mechanism Of Hypba1mentioning

confidence: 99%

“…1). HypBA1 has an exo-type activity that releases the β-Araf unit linked to the nonreducing end (14) ( …”

Section: B-2 Substrate Specificity Of Hypba1mentioning

confidence: 99%

“…HRGPs are involved in various biological processes in plants (7,8). The HRGP superfamily comprises extensin (9) and arabinogalactan-proteins (7), which are a cell-wall structural protein and a type of plant proteoglycan, respectively, as well as solanaceous lectins (10) Then, the cytosolic β-Ara 2 is hydrolyzed by HypBA1 (GH127 β-LAraf-ase) (14). HypBA1 does not have a signal peptide, thus, it is expected to be an intracellular enzyme.…”

Section: A Introductionmentioning

confidence: 99%

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Novel Active Center and Reaction Mechanism of GH127 ^|^#x3B2;-L-Arabinofuranosidase from Bifidobacterium longum subsp. longum JCM1217 (HypBA1)

Ito

2014

TIGG

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Plants have hydroxyproline-rich glycoproteins (HRGPs), containing hydroxyproline residues that are modified by β-arabinooligosaccharides. Several enzymes that catalyze the degradation of β-arabinooligosaccharides in HRGPs were recently discovered in Bifidobacterium longum JCM1217. This review describes three-dimensional structures of one of those enzymes, GH127 β-L-arabinofuranosidase (HypBA1). The X-ray crystal structures of HypBA1 in its apo form and in complex with β-Araf were determined at resolution of 2.2 Å and 2.0 Å respectively. HypBA1 was found to have a novel active center and was suggested to catalyze reactions by an unprecedented mechanism in glycosidases. The proposed reaction mechanism, which was supported by biochemical analysis, uses a Cys residue that coordinates a Zn 2+ ion as a nucleophile. There are many homologs of HypBA1 in bacteria, fungi, and plants, and the catalytic residues are highly conserved among them. Therefore, it is predicted that a substantial number of enzymes share a similar reaction mechanism with HypBA1.

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Synthesis of the Highly Glycosylated Hydrophilic Motif of Extensins

Ishiwata

Kaeothip²,

Takeda³

et al. 2014

Angewandte Chemie

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Extensin, the structural motif of plant extracellular matrix proteins, possesses a unique highly glycosylated, hydrophilic, and repeating Ser1Hyp4 pentapeptide unit, and has been proposed to include post‐translational hydroxylation at proline residue and subsequent oligo‐L‐arabinosylations at all of the resultant hydroxyprolines as well as galactosylation at serine residue. Reported herein is the stereoselective synthesis of one of the highly glycosylated motifs, Ser(Galp1)‐Hyp(Araf4)‐Hyp(Araf4)‐Hyp(Araf3)‐Hyp(Araf1). The synthesis has been completed by the application of 2‐(naphthyl)methylether‐mediated intramolecular aglycon delivery to the stereoselective construction of the Ser(Galp1) and Hyp(Arafn) fragments as the key step, as well as Fmoc solid‐phase peptide synthesis for the backbone pentapeptide.

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Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum

Cited by 53 publications

References 34 publications

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Novel Active Center and Reaction Mechanism of GH127 ^|^#x3B2;-L-Arabinofuranosidase from Bifidobacterium longum subsp. longum JCM1217 (HypBA1)

Synthesis of the Highly Glycosylated Hydrophilic Motif of Extensins

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