8IBS

Crystal structure of GH42 beta-galactosidase BiBga42A from Bifidobacterium longum subspecies infantis E160A/E318A mutant in complex with galactose

Summary for 8IBS

• Entry DOI: 10.2210/pdb8ibs/pdb
• Descriptor: Beta-galactosidase, alpha-D-galactopyranose (3 entities in total)
• Functional Keywords: glycoside hydrolase family 42, hydrolase
• Biological source: Bifidobacterium longum subsp. infantis (strain ATCC 15697 / DSM 20088 / JCM 1222 / NCTC 11817 / S12)
• Total number of polymer chains: 6
• Total formula weight: 473225.67

Authors

Hidaka, M.,Fushinobu, S.,Gotoh, A.,Katayama, T. (deposition date: 2023-02-10, release date: 2023-06-07, Last modification date: 2023-12-20)

Primary citation

Gotoh, A.,Hidaka, M.,Sakurama, H.,Nishimoto, M.,Kitaoka, M.,Sakanaka, M.,Fushinobu, S.,Katayama, T.
Substrate recognition mode of a glycoside hydrolase family 42 beta-galactosidase from Bifidobacterium longum subspecies infantis ( Bi Bga42A) revealed by crystallographic and mutational analyses.
Microbiome Res Rep, 2:20-20, 2023

PubMed Abstract: subsp. uses a glycoside hydrolase (GH) family 42 β-galactosidase (Bga42A) for hydrolyzing lacto--tetraose (LNT), which is the most abundant core structure of human milk oligosaccharides (HMOs). As such, Bga42A represents one of the pivotal enzymes underpinning the symbiosis between bifidobacteria and breastfed infants. Despite its importance, the structural basis underlying LNT hydrolysis by Bga42A is not understood. Moreover, no substrate-complexed structures are available to date for GH42 family members. X-ray crystallography was used to determine the structures of Bga42A in the apo- and liganded forms. The roles of the amino acid residues that were presumed to be involved in catalysis and substrate recognition were examined by a mutational study, in which kinetic parameters of each mutant were determined using 4-nitrophenyl-β-D-galactoside, lacto--biose I, LNT, and lacto--neotetraose (LNnT) as substrates. Conservation of those amino acid residues was examined among structure-determined GH42 β-galactosidases. Crystal structures of the wild-type enzyme complexed with glycerol, the E160A/E318A double mutant complexed with galactose (Gal), and the E318S mutant complexed with LNT were determined at 1.7, 1.9, and 2.2 Å resolutions, respectively. The LNT molecule (excluding the Gal moiety at subsite +2) bound to the E318S mutant is recognized by an extensive hydrogen bond network and several hydrophobic interactions. The non-reducing end Gal moiety of LNT adopts a slightly distorted conformation and does not overlap well with the Gal molecule bound to the E160A/E318A mutant. Twelve of the sixteen amino acid residues responsible for LNT recognition and catalysis in Bga42A are conserved among all homologs including β-1,6-1,3-galactosidase (Gal42A) from subsp. . Gal42A is active on 3-β-galactobiose similarly to Bga42A but is inactive on LNT. Interestingly, we found that the entrance of the catalytic pocket of Gal42A is narrower than that of Bga42A and seems not easily accessible from the solvent side due to the presence of two bulky amino acid side chains. The specificity difference may reflect the structural difference between the two enzymes.

PubMed: 38046823
DOI: 10.20517/mrr.2023.14

Experimental method

X-RAY DIFFRACTION (1.9 Å)