Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Bifidobacterial GH146 ß-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-ß-l-Araf-(1→2)-ß-l-Araf-(1→2)-ß-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 ß-l-arabinobiosidase (HypBA2), producing ß-l-Araf-(1→2)-l-Ara (ß-l-arabinobiose) and mono-ß-l-Araf linked to the HRGP backbone. In bifidobacteria, the ß-l-arabinobiose is then hydrolyzed by GH127 ß-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 ß-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

Substrate complex structure, active site labeling and catalytic role of the zinc ion in cysteine glycosidase.

ß-l-Arabinofuranosidase HypBA1 from Bifidobacterium longum belongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+ atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ ion and cysteine in catalysis as well as the substrate-bound structure were studied based on biochemical and crystallographic approaches. The enzymatic activity of HypBA1 decreased after dialysis in the presence of EDTA and guanidine hydrochloride and was then recovered by the addition of Zn2+. The Michaelis complex structure was determined using a crystal of a mutant at the acid/base catalyst residue (E322Q) soaked in a solution containing the substrate p-nitrophenyl-ß-l-arabinofuranoside. To investigate the covalent thioglycosyl enzyme intermediate structure, synthetic inhibitors of l-arabinofuranosyl haloacetamide derivatives with different anomer configurations were used to target the nucleophilic cysteine. In the crystal structure of HypBA1, ß-configured l-arabinofuranosylamide formed a covalent link with Cys417, whereas α-configured l-arabinofuranosylamide was linked to a noncatalytic residue Cys415. Mass spectrometric analysis indicated that Cys415 was also reactive with the probe molecule. With the ß-configured inhibitor, the arabinofuranoside moiety was correctly positioned at the subsite and the active site integrity was retained to successfully mimic the covalent intermediate state.

Mechanism-based inhibition of GH127/146 cysteine glycosidases by stereospecifically functionalized l-arabinofuranosides.

To understand the precise mechanism of the glycoside hydrolase (GH) family 127, a cysteine ß-l-arabinofuranosidase (Arafase) - HypBA1 - has been isolated from Bifidobacterium longum in the human Gut microbiota, and the design and synthesis of the mechanism-based inhibitors such as l-Araf-haloacetamides have been carried out. The α-l-Araf-azide derivative was used as the monoglycosylamine equivalent to afford the l-Araf-chloroacetamides (α/ß-1-Cl) as well as bromoacetamides (α/ß-1-Br) in highly stereoselective manner through Staudinger reaction followed by amide formation with/without anomerization. Against HypBA1, the probes 1, especially in the case of α/ß-1-Br inhibited the hydrolysis. Conformational implications of these observations are discussed in this manuscript. Additional examinations using l-Araf-azides (α/ß-5) resulted in further mechanistic observations of the GH127/146 cysteine glycosidases, including the hydrolysis of ß-5 as the substrate and oxidative inhibition by α-5 using the GH127 homologue.

Synthesis of naturally occurring ß-l-arabinofuranosyl-l-arabinofuranoside structures towards the substrate specificity evaluation of ß-l-arabinofuranosidase.

Methyl ß-l-arabinofuranosyl-(1 â†’ 2)-, -(1 â†’ 3)-, and -(1 â†’ 5)-α-l-arabinofuranosides have been stereoselectively synthesized through 2-naphthylmethyl ether-mediated intramolecular aglycon delivery (NAP-IAD), whose ß-linkages were confirmed by NMR analysis on the 3JH1-H2 coupling constant and 13C chemical shift of C1. The NAP-IAD approach was simply extended for the synthesis of trisaccharide motifs possessing ß-l-arabinofuranosyl-(1 â†’ 5)-l-arabinofuranosyl non-reducing terminal structure with the branched ß-l-arabinofuranosyl-(1 â†’ 5)-[α-l-arabinofuranosyl-(1 â†’ 3)]-α-l-arabinofuranosyl and the liner ß-l-arabinofuranosyl-(1 â†’ 5)-ß-l-arabinofuranosyl-(1 â†’ 5)-ß-l-arabinofuranosyl structures in olive arabinan and dinoflagellate polyethers, respectively. The results on the substrate specificity of a bifidobacterial ß-l-arabinofuranosidase HypBA1 using the regioisomers indicated that HypBA1 could hydrolyze all three linkages however behaved clearly less active to ß-(1 â†’ 5)-linked disaccharide than other two regioisomers including the proposed natural degradation product, ß-(1 â†’ 2)-linked one from plant extracellular matrix such as extensin. On the other hand, Xanthomonas XeHypBA1 was found to hydrolyze all three disaccharides as the substrate with higher specificity to ß-(1 â†’ 2)-linkage than bifidobacterial HypBA1.

Preparation of p-nitrophenyl ß-l-arabinofuranoside as a substrate of ß-l-arabinofuranosidase.

Synthesis of p-nitrophenyl ß-l-arabinofuranoside 1 as the substrate for novel ß-l-arabinofuranosidase has been achieved by using both our inter- and intra-molecular glycosylation methodologies. Although the intermolecular glycosylation with l-Araf donors 3 and 4 resulted in a mixture of both α- and ß-isomers, NAP ether-mediated IAD with 3 and 6 afforded the desired ß-l-arabinofuranoside stereospecifically which was confirmed by NMR analysis on the (3)JH1-H2 coupling constant and (13)C chemical shift of C1. As expected, 1 has been revealed to be an efficient substrate in the biological study of a novel ß-arabinofuranosidase such as HypBA1 with higher apparent affinity compared with other reported substrates.