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

Abstract

β-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-β-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, β-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 β-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.

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

“…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.…”

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

“…This is consistent with the structural characteristics of Bll3HypBA1, which contains no large structural element covering the active site. 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 ).…”

“…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 ).…”

β-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining β-l-Arabinofuranosidases

“…BtGH146 and HypBA1 were produced and purified according to the published procedures. 13,15 Intact Mass Spectrometry. rBtGH146 was diluted to 1 mg mL −1 in an SEC buffer.…”

“…The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.3c00558. Synthetic procedures, characterization of compounds, biochemical inhibition data, intact mass spectrometry, 1 H and 13 C NMR spectra for all newly synthesized compounds, X-ray data processing, and refinement statistics (PDF)…”