Crystal structure of the Enterococcus faecalis α‐N‐acetylgalactosaminidase, a member of the glycoside hydrolase family 31

Abstract

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Biochemical characterization of Bombyx mori α‐N‐acetylgalactosaminidase belonging to the glycoside hydrolase family 31

“…S1). These features imply that BmNag31 is not a secretory protein, thus it differs from EfNag31A, which may be secreted and works on the cell surface of E. faecalis (Miyazaki and Park, 2020).…”

“…The resulting amplicons were then ligated into pET28a vectors (Merck) using NdeI and XhoI restriction sites, and NheI and XhoI sites, respectively. The expression plasmid for EfCBM32 (residues 981–1126) was generated by inverse PCR using the EfNag31A‐harboring pET28a as a template, which was constructed in a previous study (Miyazaki and Park, 2020). The identities of all DNAs were confirmed by sequencing and the nucleotide sequence of BmNag31 was submitted to the DDBJ/EMBL/GenBank databases with the accession number LC581276.…”

“…Purified MBP‐BmNag31 and BmGH31 were concentrated to 5 mg/ml with Amicon Ultra centrifugation filter units, then the concentrated proteins were applied onto a HiLoad 16/60 Superdex 200 prep grade column and eluted by 1.2 column volumes of 20 mM sodium phosphate buffer (pH 7.0) containing 300 mM NaCl. EfNag31A and EfGH31 were also analyzed by gel filtration chromatography according to previously published procedures (Miyazaki and Park, 2020). For blue native PAGE, 1 mg/ml of each protein and marker protein solution (HMW Native Marker Kit, GE Healthcare) were individually mixed with an equal volume of sample buffer [1% (w/v) Coomassie Brilliant Blue G‐250, 20 mM 6‐aminocaproic acid, 20 mM Bis‐Tris–HCl (pH 7.0), 20% (v/v) glycerol] and then incubated on ice for 5 min.…”

“…A recent report indicates that the GH31 proteins, BpGH31 and BcGH31, from the human gut bacteria Phocaeicola plebeius (formerly Bacteroides plebeius ) and Bacteroides caccae , respectively, exhibit α‐ N ‐acetylgalactosaminidase (αGalNAcase) activity, removing the O ‐linked α‐ N ‐acetylgalactosamine (GalNAc) residue from a glycopeptide but not the blood type A antigen (Rahfeld et al ., 2019). Moreover, we recently determined the crystal structure of GH31 αGalNAcase (EfNag31A) from E. faecalis (Miyazaki and Park, 2020). The sequence of its xenolog, BmNag31, is 39.9%–52.5% identical to them.…”

“…GH31 is a large family comprising diverse enzymes with various substrate specificities and reaction mechanisms. It includes GHs such as α‐glucosidase (Kashiwabara et al ., 2000), α‐xylosidase (Lovering et al ., 2005), and α‐galactosidase (Miyazaki and Park, 2020); and it also includes transglycosidases (Aga et al ., 2002) and α‐glucan lyases (Rozeboom et al ., 2013). Aside from BmNag31, most of the other GH31 proteins encoded on the B. mori genome share homology with metabolic enzymes in animals, such as endoplasmic reticulum α‐glucosidase II and acid α‐glucosidase, which are involved in N ‐glycan processing (Watanabe et al ., 2013; D'Alessio and M. Dahms, 2015) and lysosomal degradation of glycogen (Roig‐Zamboni et al ., 2017), respectively.…”