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

Ishiwata, Akihiro; Narita, Satoru; Kimura, Kenta; Tanaka, Katsunori; Fujita, Kiyotaka; Fushinobu, Shinya; Ito, Yukishige

doi:10.1016/j.bmc.2022.117054

Cited by 7 publications

(6 citation statements)

References 62 publications

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“…The optimal temperature and pH for Bll4HypBA1 hydrolysis were estimated as 35 °C and 3.5, respectively (Figure 3). Additionally, the hydrolysis of the substrate with Bll4HypBA1 was inhibited by α/β‐ l ‐Ara f bromoacetamide [15b,c] (Figure SI‐4 and SI‐5), probably through a mechanism similar to that of Bll1HypBA1. Since Bll4HypBA1 did not show activity against any β‐ l ‐Ara f (1→2)‐ l ‐Ara f motif, Bll4HypBA1 was hypothesized to act after stepwise degradation with other bifidobacterial enzymes, such as HypAA and HypBA2.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2023

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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-, whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf 4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf 3 -linked structure. The latter is then cleaved by GH121 β-larabinobiosidase (HypBA2), producing β-l-Araf-(1!2)-l-Ara (βl-arabinobiose) and mono-β-l-Araf linked to the HRGP backbone. In bifidobacteria, the β-l-arabinobiose is then hydrolyzed by GH127 β-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 β-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf 1 -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-Araf n ) (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-Araf 1 ) motif.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2023

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“…The Zn atom of Bll3HypBA1 is coordinated by Cys 3 -Glu residues, and GH127 and GH146 enzymes share this structural characteristic (Cartmell et al 2018 ; Ito et al 2014 ; Luis et al 2018 ; Maruyama et al 2022 ; McGregor et al 2021 ). In our previous studies, we elucidated the reaction mechanism of GH127 Bll1HypBA1 using synthetic inhibitors that were specifically designed for cysteine glycosidase (Ishiwata et al 2022b ; Maruyama et al 2022 ). It has been demonstrated that the coordination of cysteine to Zn 2+ is necessary for deglycosylation from the thioglycosyl intermediate, which is energetically unfavorable in the absence of metal coordination (McGregor et al 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…For HPAEC-PAD, oligosaccharides were analyzed using a CarboPac PA-1 column (Dionex Corp., Sunnyvale, CA, USA) as described previously (Sasaki et al 2021 ). For HPLC, ABEE-labeled Ara f -β1,3-Ara f Gal 2 and reaction products were analyzed by a Cosmosil Sugar-D (Nacalai Tesque Inc.) as described previously (Ishiwata et al 2022b ).…”

Section: Methodsmentioning

confidence: 99%

“…longum JCM 1217 degrades Ara f- β1,2-Ara released from Ara 3 -Hyp by GH121 β- l -arabinobiosidase (HypBA2: BLLJ_0212) (Fujita et al 2011 ). Crystallographic studies of Bll1HypBA1 have revealed that a cysteine residue serves as the catalytic nucleophile (Ishiwata et al 2022b ; Ito et al 2014 ; Maruyama et al 2022 ; McGregor et al 2021 ). In addition to GH137 and GH142 β- l -arabinofuranosidases for RG-II degradation in Bacteroides thetaiotaomicron (Ndeh et al 2017 ), GH146 was established after characterization of BT0349 as an β- l -arabinofuranosidase for arabinan-derived arabinotetraose and β1,2- l -arabinobiose (Luis et al 2018 ).…”

Section: Introductionmentioning

confidence: 99%

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Bifidobacterial GH146 β-l-arabinofuranosidase for the removal of β1,3-l-arabinofuranosides on plant glycans

Fujita,

Tsunomachi,

Lixia

et al. 2024

Appl Microbiol Biotechnol

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l-Arabinofuranosides with β-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a β-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 β-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-β1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-β1,3-Araf structures but not those containing Araf-β1,2-Araf and Araf-β1,5-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 β-sandwich domains. A hairpin structure with two β-strands was observed in Bll3HypBA1, to extend from a β-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 β1,3-specific β-l-arabinofuranosidase. Key points • β1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • β-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a β1,3-linkage-specific β-l-arabinofuranosidase.

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“…Recently, a member of the family 172 of glycoside hydrolase (GH172) from Bifidobacterium bifidum was characterized as difructose dianhydride I synthase/hydrolase (αFFase1), so it can hydrolyze para-nitrophenyl-α-D-arabinofuranoside, and more importantly, interconvert inulobiose (β -D-Fruf -1,2-β -D-Fruf ) into DFA I. 16,17 From reference 16, we extracted the following relevant information: a) When a solution of inulobiose and αFFase1 reaches the chemical equilibrium, the inulobiose/DFA I ratio is 1:8.9.…”

Section: Introductionmentioning

confidence: 99%

GH172 difructose dianhydride I synthase: A two-stepped journey via glycosylation and cyclization using QM/MM metadynamics

Di Geronimo,

Kashima,

Ishiwata

et al. 2023

Preprint

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Difructose dianhydride I synthase/hydrolase (αFFase1) is a member of the family 172 of glycoside hydrolases which catalyzes the reversible transformation of inulobiose into difructose dianhydride I (DFA I). This transformation was hypothesized to go through a double-displacement mechanism formed by a glycosylation step and a cyclization step. During the glycosylation step, E270 protonates the O2 of the α-D-fructofuranosyl group in the subsite –1 (–1 sugar), and E291 attacks the anomeric carbon forming a glycosyl-enzyme intermediate (GEI) and releasing a water molecule. During the cyclization, activated by a complex torsional mechanism, the O1 of the β -D-fructofuranosyl group in the subsite +1 attacks the anomeric carbon, cleaving the glycosyl-E291 bond and transferring a proton to the E270. Using hybrid QM/MM metadynamics methods, we demonstrate the energetic accessibility of the glycosylation + cyclization mechanism for the αFFase1-inulobiose complex. Our simulations show a slightly exothermic global reaction (∆G 0 = –1.3 kcal·mol−1) in good agreement with the experimental inulobiose/DFA I resulting ratios (1:8.9). The ∆G ‡ and ∆G 0 for the glyco- sylation are 12.3 and –2.8 kcal·mol−1. The rate-limiting step is the cyclization with a ∆G ‡ = 15.3 kcal·mol−1. Our study shows that the –1 sugar follows the E5 → E5/4T5 → E3 and E3 → 4E → 4E conformational pathways. Further analysis of the cyclization simulation shows the role of E85, K147, and N226 stabilizing the rotation of the +1 sugar and facilitating the attack of the O1’ to the anomeric carbon.

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

Cited by 7 publications

References 62 publications

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

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

Bifidobacterial GH146 β-l-arabinofuranosidase for the removal of β1,3-l-arabinofuranosides on plant glycans

GH172 difructose dianhydride I synthase: A two-stepped journey via glycosylation and cyclization using QM/MM metadynamics

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